Pituitary tumor transforming gene (PTTG) carboxy-terminal peptides and methods of use thereof to inhibit neoplastic cellular proliferation and/or transformation

ABSTRACT

Disclosed is a method of inhibiting neoplastic cellular proliferation and/or transformation of mammalian cells, including cells of human origin, in vitro or in vivo. The inventive method involves the use of a composition containing a pituitary tumor transforming gene carboxy-terminal peptide (PTTG-C), which can be comprised in a chimeric protein, which has the ability to regulate endogenous pituitary tumor transforming gene (PTTG) expression and/or function in a dominant negative manner. Kits comprising the inventive compositions are also disclosed for the treatment of neoplastic cellular proliferation in vitro or in vivo. Isolated PTTG-C peptides and PTTG-C-containing chimeric proteins are described.

This application is a division of U.S. patent application Ser. No.09/569,956, filed on May 12, 2000, which is a continuation-in-part ofU.S. patent application Ser. No. 08/894,251, now U.S. Pat. No.6,455,305, filed on Jul. 23, 1999, as a national stage application,under 35 U.S.C. § 371, of international application PCT/US97/21463,filed Nov. 21, 1997, which claims the priority of the filing date ofU.S. provisional patent application Ser. No. 60/031,338, filed Nov. 21,1996.

The U.S. Government has a paid-up license in this invention and theright in limited circumstances to require the patent owner to licenseothers on reasonable terms as provided for by the terms of ContractCA75979, awarded by the National Cancer Institute of the NationalInstitutes of Health.

BACKGROUND OF THE INVENTION

Throughout the application various publications are referenced inparentheses. The disclosures of these publications in their entiretiesare hereby incorporated by reference in the application in order to morefully describe the state of the art to which this invention pertains.

1. Field of the Invention

The present invention relates to a method of inhibiting neoplasticcellular proliferation and/or transformation of mammalian cells, invitro and in vivo.

2. Related Art

Neoplasms, including cancers and other tumors, are the second mostprevalent cause of death in the United States, causing 450,000 deathsper year. One in three Americans will develop cancer, and one in fivewill die of cancer (Scientific American Medicine, part 12, I, 1, sectiondated 1987). While substantial progress has been made in identifyingsome of the likely environmental and hereditary causes of cancer, thestatistics for the cancer death rate indicates a need for substantialimprovement in the therapy for cancer and related diseases anddisorders.

A number of cancer genes, i.e., genes that have been implicated in theetiology of cancer, have been identified in connection with hereditaryforms of cancer and in a large number of well-studied tumor cells.Studies of cancer genes have helped provide some understanding of theprocess of tumorigenesis. While a great deal more remains to be learnedabout cancer genes, the presently known cancer genes serve as usefulmodels for understanding tumorigenesis.

Cancer genes are broadly classified into “oncogenes” which, whenactivated, promote tumorigenesis, and “tumor suppressor genes” which,when damaged, fail to suppress tumorigenesis. While theseclassifications provide a useful method for conceptualizingtumorigenesis, it is also possible that a particular gene may playdiffering roles depending upon the particular allelic form of that gene,its regulatory elements, the genetic background and the tissueenvironment in which it is operating.

More than 100 oncogenes have been discovered, but only a smallpercentage appear mutated in tumors. (Bishop, J. M., Molecular themes inoncogenesis, Cell 64:235-248 [1991]; Sager, R., Expression genetics incancer: shifting the focus from DNA to XNA, Proc. Natl. Acad Sci.94:952-955 [1997]). Most cancer-related genes exhibit altered expressionpatterns (increasing or decreasing), causing phenotypic changesinvolving signal transduction, cell proliferation, DNA repair,angiogenesis, and apoptosis. (Pawson, T., and Hunter, T., Signaltransduction and growth control in normal and cancer cells, Curr. OpinGene Dev. 4:1-4 [1994]; Bartek, J., et al., Defects in cell cyclecontrol and cancer, J Pathol, 187:95-99 [1999]; Sancer, A., Mechanismsof DNA excision repair, Science 266:1954-1956 [1994]; Hanahan, D., andFolkman, J., Pattern and emerging mechanisms of the angiogenic switchduring tumorigenesis, Cell 86:353-346 [1996]; Wyllie, A. H., The geneticregulation of apoptosis, Curr, Opin. Gene Dev. 5:97-104 [1995]).Identifying specific regulators modulating oncogene expression isimportant to provide the basis for development of potential subcellulartherapeutic strategies. (Gibbs, J. B., and Oliff A., Pharmaceuticalresearch in molecular oncology, Cell 79:193-198 [1994]; Levitzki A.,Signal-transduction therapy: a novel approach to disease management,Eur. J. Biochem. 226:1-13 [1994]).

Tumor suppressor genes play a role in regulating oncogenesis. Tumorsuppressor genes are genes that in their wild-type alleles, expressproteins that suppress abnormal cellular proliferation. When the genecoding for a tumor suppressor protein is mutated or deleted, theresulting mutant protein or the complete lack of tumor suppressorprotein expression may fail to correctly regulate cellularproliferation, and abnormal cellular proliferation may take place,particularly if there is already existing damage to the cellularregulatory mechanism. A number of well-studied human tumors and tumorcell lines have been shown to have missing or nonfunctional tumorsuppressor genes. Examples of tumor suppressor genes include, but arenot limited to the retinoblastoma susceptibility gene or RB gene, thep53 gene, the deleted in colon carcinoma (DDC) gene and theneurofibromatosis type 1 (NF-1) tumor suppressor gene (Weinberg, R. A.,Science, 254:1138-46 [1991]). Loss of function or inactivation of tumorsuppressor genes may play a central role in the initiation and/orprogression of a significant number of human cancers.

Anterior pituitary tumors are mostly benign hormone-secreting ornon-functioning adenomas arising from a monoclonal expansion of agenetically mutated pituitary epithelial cell. Pathogenesis of tumorformation in the anterior pituitary has been intensively studied.Mechanisms for pituitary tumorigenesis involve a multi-step cascade ofrecently characterized molecular events. The most well characterizedoncogene in pituitary tumors is gsp; a constitutively activated G(s)αprotein results from certain point mutations in gsp. (E.g., Fragoso, M.C., et al., Activating mutation of the stimulatory G protein [gsp] as aputative cause of ovarian and testicular human stromal Leydig celltumors, J. Clin. Endocrinol. 83(6):2074-78 [1999]; Barlier, A. et al.,Impact of gsp oncogene on the expression of genes coding for Gsalpha,Pit-I, Gi2alpha, and somatostatin receptor 2 in human somatotrophadenomas: involvement of octreotide sensitivity, J. Clin. Endocrinol.Metab. 84(8):2759-65 [1999]; Ballare, E., et al., Activating mutationsof the Gs alpha gene are associated with low levels of Gs alpha proteinin growth hormone-secreting tumors, J. Clin. Endocrinol. Metab.83(12):4386-90 [1999]).

G(s)α mutations occur in about 40% of growth hormone (GH)-secretingtumors, and constitutively activated CREB transcription factor is alsofound in a subset of these tumors. Although the importance of GSα mutantproteins in the development of growth-hormone secreting pituitary tumorsis well established, only about one third of these tumors contains thesemutations, indicating the presence of additional transforming events inpituitary tumorigenesis. Although point mutations of Ras oncogene, lossof heterozygosity (LOH) near the Rb locus on chromosome 13, and LOH onchromosome 11 have been implicated in some pituitary tumors, themechanism that causes pituitary cell transformation remains largelyunknown.

Recently, a novel pituitary tumor transforming gene, PTTG (previouslyknown as pituitary-tumor-specific-gene [PTSG]), was isolated. PTTGencodes a securin protein the expression of which causes celltransformation, induces the production of basic fibroblast growth factor(bFGF), is regulated in vitro and in vivo by estrogen, and inhibitschromatid separation. (Pei, L., and Melmed S., Isolation andcharacterization of a pituitary tumor transforming gene, Mol.Endocrinol. 11:433-441 [1997]; Zhang, X., et al., Structure, expression,and function of human pituitary tumor-transforming gene (PTTG), Mol.Endocrinol. 13:156-166 [1999a]; Heaney, A. P., et al., Early involvementof estrogen-induced pituitary tumor transforming gene and fibroblastgrowth factor expression in prolactinoma pathogenesis, Nature Med.5:1317-1321 [1999]; Zou, H., et al., Identification of a vertebratesister-chromatid separation inhibitor involved in transformation andtumorigenesis, Science 285:418-422 [1999]).

By dysregulating chromatid separation, PTTG overexpression may also leadto aneuploidy, i.e., cells having one or a few chromosomes above orbelow the normal chromosome number (Zou et al. [1999]). Like mostcancer-related genes, the expression of PTTG is restricted in normaltissues, but PTTG expression is dramatically increased in malignanthuman cell lines, pituitary tumors, colon carcinomas and colorectaltumors. (Zhang, X., et al. [1999a]; Zhang, X., et al., Pituitary tumortransforming gene (PTTG) expression in pituitary adenornas, J. Clin.Endocrinol. Metab. 84:761-767 [1999b]; Heaney, A. R., et al., Pituitarytumor transforming gene: a novel marker in colorectal tumors, Lancet [InPress; 2000]).

The recent discovery of a human PTTG gene 2, which shares high sequencehomology with human PTTG1, implying the existence of a PTTG gene family.(Prezant, T. R., et al., An intronless homolog of human proto-oncogenehPTTG is expressed in pituitary tumors: evidence for hPTTG family, J.Clin. Endocrinol. Metab. 84:1149-1152 [1999]). Murine PTTG shares 66%nucleotide base sequence homology with human PTTG1 and also exhibitstransforming ability. (Wang, Z. and Melmed, S., Characterization of themurine pituitary tumor transforming gone (PTTG) and its promoter,Endocrinology [In Press; 2000]). A proline-rich region was identifiednear the protein C-terminus that is critical for PTTG1's transformingactivity. (Zhang, X., et al. [1999a]), as demonstrated by the inhibitoryeffect on in vitro transformation, in vivo tumorigenesis, andtransactivation, when point mutations were introduced into theproline-rich region. Proline-rich domains may function as SH3 bindingsites to mediate signal transduction of protein-tyrosine kinase.(Pawson, T., Protein modules and signaling networks, Nature 373:573-580[1995]; Kuriyan, J., and Cowburn, D., Modular peptide recognitiondomains in eukaryotic signaling, Annu, Rev. Biophys. Biomol. Struct.26:259-288 [1997]).

There remains a need for a therapeutic treatment for neoplasms, such ascancer, that inhibits neoplastic cellular proliferation and/ortransformation associated with PTTG overexpression. This and otherbenefits are provided by the present invention as described herein.

SUMMARY OF THE INVENTION

The present invention relates to a method of inhibiting neoplasticcellular proliferation and/or transformation of mammalian cells,including cells of human origin, whether in vitro or in vivo. Theinventive method relies on the discovery that the nativecarboxy-terminal portion of the pituitary tumor transforming geneprotein (PTTG) is critical to PTTG protein function and that,surprisingly, pituitary tumor transforming gene carboxy-terminal peptide(PTTG-C) molecules have the ability to downregulate pituitary tumortransforming gene (PTTG) expression and/or PTTG function in a dominantnegative manner. In some embodiments, the invention is directed togene-based treatments that deliver PTTG carboxy-terminal-relatedpolynucleotides to mammalian cells to inhibit the endogenous expressionand function of PTTG. Other embodiments are directed to peptide-basedtreatments that deliver PTTG-C peptides to the cells, which inhibitendogenous PTTG expression and/or PTTG function.

In particular, useful gene-based embodiments of the method of inhibitingneoplastic cellular proliferation and/or transformation of mammaliancells involve delivering to the cell a composition comprising aPTTG-C-related polynucleotide that includes a base sequence that definesa PTTG carboxy-terminal peptide-encoding sequence, or defines adegenerate sequence, or defines a sequence complementary to either ofthese. In accordance with the method, the PTTG carboxy-terminal-relatedpolynucleotide, preferably complexed with a cellular uptake-enhancingagent, is delivered in an amount and under conditions sufficient toenter the cell, thereby inhibiting neoplastic cellular proliferationand/or transformation of the cell.

Alternatively, useful peptide-based embodiments of the method ofinhibiting neoplastic cellular proliferation and/or transformation of amammalian cell involve delivering to a mammalian cell a compositioncomprising a PTTG carboxy-terminal peptide (PTTG-C), or a biologicallyfunctional fragment thereof, preferably complexed with a cellularuptake-enhancing agent, in an amount and under conditions sufficient toenter the cell, thereby inhibiting neoplastic cellular proliferationand/or transformation.

Because, PTTG protein further mediates the expression of bFGF, animportant angiogenesis activator, the inventive method of inhibitingneoplastic cellular proliferation and/or transformation, practiced invivo, also encompasses a method of inhibiting tumor angiogenesis.Angiogenesis activators, including bFGF and VEGF, are expressed andsecreted by most human carcinoma cells. (Plate, K. H. et al., Nature359:845-48 [1992]; Schultz-Hector, S. and Haghayegh, S., Cancer Res.53:1444-49 [1993];Yamanaka, Y. et al., Cancer Res. 53:5289-96 [1993];Buensing, S. et al., Anticancer Res. 15:2331-34 [1995]). The discovery,described herein, that the inventive PTTG-C peptides dramatically reducebFGF production by cancer cells (e.g., HeLa), shows that in accordancewith the inventive method, the inventive PTTG-C peptides can impair newblood vessel growth, which is essential for tumor growth. Thus, themethod of inhibiting tumor angiogenesis further inhibits neoplasticcellular proliferation, in vivo.

The present invention also relates to compositions useful for inhibitingneoplastic cellular proliferation and/or transformation. These includecompositions comprising a PTTG carboxy-terminal peptide or comprising achimeric or fusion protein that contains a first PTTG carboxy-terminalpeptide segment and a second cellular uptake-enhancing peptide segment.The invention also relates to compositions comprising a PTTGcarboxy-terminal-related polynucleotide, for example, a polynucleotideencoding a PTTG-C peptide or antisense PTTG-C-related oligonucleotides.Also included in the invention are compositions comprising expressionvectors containing the PTTG-C-related polynucleotides, including nucleicacids encoding PTTG-C peptides. The inventive PTTG-C peptides andinventive PTTG-C-related polynucleotides are useful in the manufactureof pharmaceutical compositions, medicaments or medicants for inhibitingneoplastic cellular proliferation and/or transformation, which containthe inventive PTTG-C peptides and PTTG-C-related polynucleotides.

In accordance with the present invention, there are also provided PTTGcarboxy-terminal (PTTG-C) peptides and PTTG-C-related polynucleotides,which can also be isolated from other cellular components. The inventivePTTG-C peptides are useful in bioassays, as immunogens for producinganti-PTTG antibodies, or in therapeutic compositions containing suchpeptides and/or antibodies. Also provided are transgenic non-humanmammals that comprise mammalian cells that comprise embodiments of theinventive PTTG-C-related polynucleotides and express the inventivePTTG-C peptides.

Also provided are antibodies that are specifically immunoreactive withPTTG proteins, or more particularly, with PTTG-C peptides. The inventiveantibodies specifically bind to PTTG-C peptides. Theseanti-PTTG-C-specific antibodies are useful in assays to determine levelsof PTTG proteins or PTTG-C peptides present in a given sample, e.g.,tissue samples, biological fluids, Western blots, and the like. Theantibodies can also be used to purify PTTG proteins or PTTG-C peptidesfrom crude cell extracts and the like. Moreover, these antibodies areconsidered therapeutically useful to counteract or supplement thebiological effect of PTTG proteins in vivo.

The present invention is further described by related applications U.S.Ser. No. 08/894,251, filed Jul. 23, 1999, international applicationPCT/US97/21463, filed Nov. 21, 1997, and U.S. provisional application60/031,338, filed Nov. 21, 1996, the disclosures of which areincorporated by reference,

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates transcriptional activation in transfected NIH 3T3cells, as mediated by pGAL4, pGAL4-VP16, pGAL4-wtPTTG, orpGAL4-mutPTTG48 hours after transfection. Cell lysate proteins wereassayed for luciferase and β-gal expression. pGAL4 was used as anegative control and pGAL4-VP16 as a positive control.

FIG. 2 shows PTTG-C and PTTG-Cpm expression in transfected tumor cells.FIG. 2A illustrates expression construct which express a C-terminalpeptide of human PTTG protein (PTTGC), corresponding to amino acidresidues 147-202 of SEQ. ID. NO.:4 (i.e., SEQ. ID. NO.:9), under thecontrol of the CMV promoter (black bar). PXXP represents theproline-rich region(s) of the PTTG-C. A mutant expression vector(PTTG-Cpm), contained point mutations P163A, S165Q, P166L, P170L, P172A,and P173L. FIG. 2B includes representative 1% agarose gels of RT-PCRproducts of HeLa (top panel), T-47D (middle panel), and MCF-7 cells(bottom panel), showing PTTG-C and PTTG-Cpm expression. Products fromreverse transcription carried out in the presence (+) or absence (−) ofRT were used as template in PCR reactions. FIG. 2C shows arepresentative sequencing gel from RT-PCR followed by direct sequencinganalysis showing PTTG-C and PTTG-Cpm expression in respectivetransfectants. Arrows point to nucleotide changes.

FIG. 3 shows colony formation of HeLa (top row), T-47D (middle row), andMCF-7 (bottom row) cells transfected with PTTG-C or PTTG-Cpm expressionvectors on soft agar. “Vector” (left column)shows cells transfected withvector pCI-neo alone; “PTTG-C” (middle column) shows cells transfectedwith vector pCI-neo containing PTTG C-terminal encoding cDNA; “PTTG-Cpm”(right column) shows cells transfected with vector pCI-neo containingmutant PTTG C-terminal cDNA (P163A, S165Q, P166L, P170L, P172A, and P173L).

FIG. 4 shows suppression of bFGF secretion by HeLa cells expressingPTTG-C peptide. The concentration of bFGF in conditioned medium derivedfrom transfectants cultured for 72 h as measure by ELISA. “Vector” (twoleft-most bars) indicates medium conditioned by cells transfected withvector pCI-neo alone; “PTTG-C” (three middle bars) indicates mediumconditioned by cells transfected with vector pCI-neo containingwtPTTG-C-terminal encoding cDNA; “PTTG-Cpm” (three right-most bars)indicates medium conditioned by transfected with vector pCI-neocontaining mutant PTTG C-terminal encoding cDNA (P163A, S165Q, P166L,P170L, P172A, and P173L).

DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

The present invention relates to a method of inhibiting PTTG-mediatedneoplastic cellular proliferation and/or transformation of a mammaliancell, including but not limited to, a cell of human or non-human origin.Non-human mammalian cells also originate from, or in, any mammaliananimal, e.g., a non-human primate, rat, mouse, rabbit, guinea pig,hamster, bovine, porcine, ovine, equine, canine, feline, pachyderm, andthe like. The mammalian cell can be situated in vivo, i.e., within amammalian animal subject or human subject, or in vitro, i.e., the cellcan be a cultured cell.

For the purposes of the invention, “neoplastic cellular proliferation”includes neoplastic (malignant or benign), hyperplastic, cytologicallydysplastic and/or premalignant cellular growth or proliferation in amammalian subject or cell culture. Hyperplastic cellular growth orproliferation includes abnormal multiplication or increase in thenumbers of normal cells in a normal arrangement in a tissue, forexample, as is common in benign prostatic hyperplasia. Cytologicallydysplastic and/or premalignant cellular growth or proliferation includeincreases in cellular numbers of karyotypically abnormal butnon-malignant cells within a tissue. Examples include some benignprostatic hyperplasias/dysplasia and cervical hyperplasias/dysplasias.

Neoplastic cellular growth and/or proliferation, i.e., growth ofabnormally organized tissue, includes malignant and non-malignantneoplasms. Malignant neoplasms include primary, recurrent, and/or ormetastatic cancerous tumors originating in any tissues, for example,carcinomas, sarcomas, lymphomas, mesotheliomas, melanomas, gliomas,nephroblastomas, glioblastomas, oligodendrogliomas, astrocytomas,ependymomas, primitive neuroectodermal tumors, atypical meningiomas,malignant meningiomas, or neuroblastomas, originating in the pituitary,hypothalamus, lung, kidney, adrenal, ureter, bladder, urethra, breast,prostate, testis, skull, brain, spine, thorax, peritoneum, ovary,uterus, stomach, liver, bowel, colon, rectum, bone, lymphatic system,skin, or in any other organ or tissue of the subject.

In accordance with gene-based embodiments of the method of inhibitingneoplastic cellular proliferation and/or transformation, an inventivecomposition is delivered to the cell, which composition comprises a PTTGcarboxy-terminal-related polynucleotide. A “PTTGcarboxy-terminal-related” polynucleotide is a polynucleotide having acontiguous sequence of bases (e.g., adenine [A], thymine [T], uracil[U], guanine [G], and/or cytosine [C]) defining a sequence specific tothe 3′ coding region of PTTG. The 3′-end or terminal extends fromapproximately the mid-point of a cDNA coding sequence encoding a nativePTTG to its end at a stop codon. The PTTG carboxy-terminal-relatedpolynucleotide can be a sequence encoding a carboxy-terminal portion ofa mammalian PTTG protein (i.e., a PTTG-C peptide), as described morefully below, or encoding a PTTG-specific fragment thereof, or adegenerate coding sequence, or a sequence complementary to any of these.

In some preferred embodiments, the inventive composition includes anucleic acid construct, such as a plasmid or viral expression vector,which comprises the polynucleotide in a sense or antisense orientation,and from which PTTG-specific mRNA transcript can be expressed in thecell. In a preferred embodiment, the nucleic acid construct contains apolynucleotide encoding a mammalian PTTG carboxy-terminal (PTTG-C)peptide, which can be any PTTG-C peptide or functional fragment thereofas described herein. The composition can also contain one or more helperplasmids or viruses, if appropriate. The plasmid or viral expressionvector is a nucleic acid construct that includes a promoter regionoperatively linked to the polynucleotide in a transcriptional unit.

As used herein, a promoter region refers to a segment of DNA thatcontrols transcription of a DNA to which it is operatively linked. Thepromoter region includes specific sequences that are sufficient for RNApolymerase recognition, binding and transcription initiation. Inaddition, the promoter region includes sequences that modulate thisrecognition, binding and transcription initiation activity of RNApolymerase. These sequences may be cis acting or may be responsive totrans acting factors. Promoters, depending upon the nature of theregulation, may be constitutive or regulated. Exemplary promoterscontemplated for use in the practice of the present invention includethe SV40 early promoter, the cytomegalovirus (CMV) promoter, the mousemammary tumor virus (MMTV) steroid-inducible promoter, Moloney murineleukemia virus (MMLV) promoter, and the like.

As used herein, “expression” refers to the process by which polynucleicacids are transcribed into mRNA and translated into peptides,polypeptides, or proteins. If the polynucleic acid is derived fromgenomic DNA, expression may, if an appropriate eukaryotic host cell ororganism is selected, include splicing of the mRNA.

As used herein, the term “operatively linked” refers to the functionalrelationship of DNA with regulatory and effector nucleotide sequences,such as promoters, enhancers, transcriptional and translational stopsites, and other signal sequences. For example, operative linkage of DNAto a promoter refers to the physical and functional relationship betweenthe DNA and the promoter such that the transcription of such DNA isinitiated from the promoter by an RNA polymerase that specificallyrecognizes, binds to and transcribes the DNA. Thus, “operatively linked”means that, within a transcriptional unit, the promoter sequence, islocated upstream (i.e., 5′ in relation thereto) from the coding sequenceand the coding sequence, is 3′ to the promoter, or alternatively is in asequence of genes or open reading frames 3′ to the promoter andexpression is coordinately regulated thereby. Both the promoter andcoding sequences are oriented in a 5′ to 3′ manner, such thattranscription can take place in vitro in the presence of all essentialenzymes, transcription factors, co-factors, activators, and reactants,under favorable physical conditions, e.g., suitable pH and temperature.This does not mean that, in any particular cell, conditions will favortranscription. For example, transcription from a tissue-specificpromoter is generally not favored in heterologous cell types fromdifferent tissues.

The term “nucleic acid” encompasses ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA), which DNA can be complementary DNA (cDNA)or genomic DNA, e.g. a gene encoding a PTTG protein. “Polynucleotides”encompass nucleic acids containing a “backbone” formed by phosphodiesterlinkages between ribosyl or deoxyribosyl moieties. Polynucleotides alsoinclude nucleic acid analogs, for example polynucleotides havingalternative linkages as known in the art. Examples includephosphorothioate linkages (e.g., phosphorothioate oligodeoxynucleotides;S-oligonucleotides), mixed phosphorothioate and phosphodiester linkages(e.g., S-O-oligodeoxynucleotides and phosphodiester/phosphorothioate2′-O-methyl-oligoribonucleotides; Zhou, W. et al., Mixed backboneoligonucleotides as second-generation antisense agents with reducedphosphthioate-related side effects, Bioorg. Med. Chem. Lett.8(22):3269-74 [1998]), methylphosphonate-phosphodiester modifications(MP-O-oligonucleotides; Zhao, Q. et al., Comparison of cellular bindingand uptake of antisense phosphodiester, phosphorothioate, and mixedphosphorothioate and methylphosphonate oligonucleotides, Antisense Res.Dev. 3(1):53-66 [1993]), or morpholino oligonucleotides (e.g., Schmajuk,G. et al., Antisense oligonucleotides with different backbones.Modification of splicing pathways and efficacy of uptake, J. Biol. Chem.274(31):21783-89 [1999]).

Also included among useful polynucleotides are nucleic acid analogshaving a pseudopeptide or polyamide backbone comprisingN-(2-aminoethyl)glycine moieties, i.e., peptide nucleic acids (PNA).(E.g., Nielsen, P. E., Peptide nucleic acids: on the road to new genetherapeutic drugs, Pharmacol. Toxicol. 86(1):3-7 [2000]; Soomets, U. etal., Antisense properties of peptide nucleic acids, Front. Biosci.4:D782-86 [1999]; Tyler, B. M. et al., Peptide nucleic acids targeted tothe neurotensin receptor and administered i.p. cross the blood-brainbarrier and specifically reduce gene expression, Proc. Natl. Acad. Sci.USA 96(12):7053-58 [1999]).

Polynucleotides include sense or antisense polynucleotides.“Polynucleotides” also encompasses “oligonucleotides”.

A polynucleotide sequence complementary to a PTTG-specificpolynucleotide sequence, as used herein, is one binding specificallywith a PTTG-specific nucleotide base sequence. The phrase “bindingspecifically” encompasses the ability of a polynucleotide sequence torecognize a complementary base sequence and to form double-helicalsegments therewith via the formation of hydrogen bonds between thecomplementary base pairs. Thus, a complementary sequence includes, forexample, an antisense sequence with respect to a sense sequence orcoding sequence.

In some embodiments of the PTTG-C-related polynucleotide, thepolynucleotide is in a sense orientation within the transcriptionalunit, such that mRNA transcript can be produced, which when translatedresults in a translation product, such as a PTTG protein or a PTTGcarboxy-terminal peptide (PTTG-C). In other embodiments, thePTTG-C-related polynucleotide is in an antisense orientation such thattranscription results in a transcript complementary to and hybridizablewith a naturally-occurring sense PTTG mRNA molecule under physiologicalconditions, inhibiting or blocking translation therefrom. Thus,antisense oligonucleotides inactivate target mRNA sequences by eitherbinding thereto and inducing degradation of the mRNA by, for example,RNase I digestion, or inhibiting translation of mRNA target sequence byinterfering with the binding of translation-regulating factors orribosomes, or by inclusion of other chemical structures, such asribozyme sequences or reactive chemical groups which either degrade orchemically modify the target mRNA. For example, an antisenseoligonucleotide targeted to a PTTG carboxy-terminal-relatedpolynucleotide segment of mRNA or genomic DNA is effective in inhibitingexpression of PTTG.

Gene-based therapy strategies employing antisense oligonucleotides arewell known in the art. (E.g., Rait, A. et al., 3′-End conjugates ofminimally phosphorothioate-protected oligonucleotides with1-O-hexadecylglycerol: synthesis and anti-ras activity inradiation-resistant cells, Bioconjug Chem., 11(2):153-60 [2000];Stenton, G. R. et al., Aerosolized syk antisense suppresses sykexpression, mediator release from macrophages, and pulmonaryinflammation, J. Immunol., 164(7):3790-7 [2000]; Suzuki, J. et al.,Antisense Bc1-x oligonucleotide induces apoptosis and prevents arterialneointimal formation in murine cardiac allografts, Cardiovas. Res.,45(3):783-7 [2000]; Kim, J. W. et al., Antisense oligodeoxynucleotide ofglyceraldehyde-3-phosphate dehyrdogenase gene inhibits cellproliferation and induces apoptosis in human cervical carcinoma cellline, Antisense Nucleic Acid Drug Dev., 9(6):507-13 [1999]; Han, D. C.et al., Therapy with antisense TGF-betal oligodeoxynucleotides reduceskidney weight and matrix mRNAs in diabetic mice, Am. J. Physiol. RenalPhysiol., 278(4):F628-F634 [2000]; Scala, S. et al., Adenovirus-mediatedsuppression of HMGI (Y) protein synthesis as potential therapy of humanmalignant neoplasias, Proc. Natl. Acad. Sci. USA., 97(8):4256-4261[2000]; Arteaga, C. L., et al., Tissue-targeted antisense c-fosretroviral vector inhibits established breast cancer xenografts in nudemice, Cancer Res., 56(5):1098-1103 [1996]; Muller, M. et al., Antisensephosphorothioate oligodeoxynucleotide down-regulation of theinsulin-like growth factor I receptor in ovarian cancer cells, Int. J.Cancer, 77(4):567-71 [1998]; Engelhard, H. H., AntisenseOligodeoxynucleotide Technology: Potential Use for the Treatment ofMalignant Brain Tumors, Cancer Control, 5(2):163-170 [1998];Alvarez-Salas, L. M. et al., Growth inhibition of cervical tumor cellsby antisense oligodeoxynucleotides directed to the human papillomavirustype 16 E6 gene, Antisense Nucleic Acid Drug Dev., 9(5):441-50 [1999];Im, S. A., et al., Antiangiogenesis treatment for gliomas: transfer ofantisense-vascular endothelial growth factor inhibits tumor growth invivo, Cancer Res., 59(4):895-900 [1999]; Maeshima, Y. et al., Antisenseoligonucleotides to proliferating cell nuclear antigen and Ki-67 inhibithuman mesangial cell proliferation, J. Am. Soc. Nephrol., 7(10):2219-29[1996]; Chen, D. S. et al., Retroviral Vector-mediated transfer of anantisense cyclin G1 construct inhibits osteosarcoma tumor growth in nudemice, Hum. Gene Ther, 8(14):1667-74 [1997]; Hirao, T. et al., Antisenseepidermal growth factor receptor delivered by adenoviral vector blockstumor growth in human gastric cancer, Cancer Gene Ther. 6(5):423-7[1999]; Wang, X. Y. et al., Antisense inhibition of protein kinaseCalpha reverses the transformed phenotype in human lung carcinoma cells,Exp. Cell Res., 250(1):253-63 [1999]; Sacco, M. G. et al., In vitro andin vivo antisense-mediated growth inhibition of a mammary adenocarcinomafrom MMTV-neu transgenic mice, Gene Ther., 5(3);388-93 [1998]; Leonetti,C. et al., Antitumor effect of c-myc antisense phosphorothioateoligodeoxynucleotides on human melanoma cells in vitro and in mice, J.Natl. Cancer Inst., 88(7):419-29 [1996]; Laird, A. D. et al., Inhibitionof tumor growth in liver epthelial cells transfected with a transforminggrowth factor alpha antisense gene, Cancer Res. 54(15):4224-32 (Aug. 1,1994); Yazaki, T. et al., Treatment of glioblastoma U-87 by systemicadministration of an antisense protein kinase C-alpha phosphorothioateoligodeoxynucleotide, Mol. Pharmacol., 50(2):236-42 [1996]; Ho, P. T. etal., Antisense oligonucleotides as therapeutics for malignant diseases,Semin. Oncol., 24(2):187-202 [1997]; Muller, M. et al., Antisensephosphorothioate oligodeoxynucleotide down-regulation of theinsulin-like growth factor I receptor in ovarian cancer cells, Int. J.Cancer, 77(4):567-71 [1998]; Elez, R. et al., Polo-like kinasel, a newtarget for antisense tumor therapy, Biochem. Biophys. Res. Commun.,269(2):352-6 [2000]; Monia, B. P. et al., Antitumor activity of aphosphorothioate antisense oligodeoxynucleotide targeted against C-rafkinase, Nat. Med., 2(6):668-75 [1996]).

In other embodiments of the inventive method, the inventive compositioncomprises a PTTG carboxy-terminal-related polynucleotide that is notcontained in an expression vector, for example, a synthetic antisenseoligonucleotide, such as a phosphorothioate oligodeoxynucleotide.Synthetic antisense oligonucleotides, or other antisense chemicalstructures designed to recognize and selectively bind to mRNA, areconstructed to be complementary to portions of the PTTG coding strand,for example, to coding sequences shown in SEQ ID NOS:1, 3, 10, 15, 18,or 19 (Tables 1-6 below). By preventing translational expression of atleast part of the PTTG3′ coding region, an antisense PTTGcarboxy-terminal-related polynucleotide is useful, in accordance withthe inventive method, to prevent expression of PTTG protein that isfunctional in mediating neoplastic cellular proliferation and/ortransformation.

In preferred embodiments of the method of inhibiting neoplastic cellularproliferation and/or transformation, the composition also comprises anuptake-enhancing agent as further described herein. Inventivecompositions, containing the uptake-enhancing agent complexed with aPTTG-specific polynucleotide, are designed to be capable of passingthrough the cell membrane in order to enter the cytoplasm of the cell byvirtue of physical and chemical properties. In addition, the compositioncan be designed for delivery only to certain selected cell populationsby targeting the composition to be recognized by specific cellularuptake mechanisms which take up the PTTG-specific polynucleotides onlywithin select cell populations. For example, the composition can includea receptor agonist to bind to a receptor found only in a certain celltype.

The inventive composition can also optionally contain one or morepharmaceutically acceptable carrier(s). As used herein, the term“acceptable carrier” encompasses any of the standard pharmaceuticalcarriers. The carrier can be an organic or inorganic carrier orexcipient, such as water and emulsions such as an oil/water or water/oilemulsion, and various types of wetting agents. The active ingredient(s)can optionally be compounded in a composition formulated, for example,with non-toxic, pharmaceutically acceptable carriers for infusions,tablets, pellets, capsules, solutions, emulsions, suspensions, and anyother form suitable for use. Such carriers also include glucose,lactose, gum acacia, gelatin, mannitol, starch paste, magnesiumtrisilicate, talc, corn starch, keratin, colloidal silica, potatostarch, urea, medium chain length triglycerides, dextrans, normalsaline, phosphate buffered saline and other carriers suitable for use inmanufacturing preparations, in solid, semisolid, or liquid form. Inaddition auxiliary, stabilizing, thickening and coloring agents andperfumes can be used as appropriate.

PTTG-specific polynucleotides, including PTTG carboxy-terminal-relatedpolynucleotides, are determined by base sequence similarity or homologyto known mammalian PTTG-specific nucleotide sequences. Base sequencehomology is determined by conducting a base sequence similarity searchof a genomics data base, such as the GenBank database of the NationalCenter for Biotechnology Information (NCBI), using a computerizedalgorithm, such as PowerBLAST, QBLAST, PSI-BLAST, PHI-BLAST, gapped orungapped BLAST, or the “Align” program through the Baylor College ofMedicine server. (E.g., Altchul, S. F., et al., Gapped BLAST andPSI-BLAST: a new generation of protein database search programs, NucleicAcids Res. 25(17):3389-402 [1997]; Zhang, J., & Madden, T. L.,PowerBLAST: a new network BLAST application for interactive or automatedsequence analysis and annotation, Genome Res. 7(6):649-56 [1997];Madden, T. L., et al., Applications of network BLAST server, MethodsEnzymol. 266:131-41 [1996]; Altschul, S. F., et al., Basic localalignment search tool, J. Mol. Biol. 215(3):403-10 [1990]). Preferably,a PTTG-specific polynucleotide sequence is at least 5 to 30 contiguousnucleotides long, more preferably at least 6 to 15 contiguousnucleotides long, and most preferably at least 7 to 10 contiguousnucleotides long. Preferably, the inventive PTTGcarboxy-terminal-related polynucleotide is at least about 45 contiguousnucleotides long.

Preferred examples of PTTG-specific coding sequences include thesequence for human PTTG (hPTTG or PTTG1). The PTTG1 peptide is encodedby the open reading frame at nucleotide positions 95 through 700 ofhuman PTTG1 gene sequence SEQ. ID. NO.:3 (Table 1 below).

TABLE 1 PTTG1 gene sequence. 1 ATGGCCGCGA GTTGTGGTTT AAACCAGGAGTGCCGCGCGT CCGTTCACCG 51 CGGCCTCAGA TGAATGCGGC TGTTAAGACC TGCAATAATCCAGAATGGCT 101 ACTCTGATCT ATGTTGATAA GGAAAATGGA GAACCAGGCA CCCGTGTGGT151 TGCTAAGGAT GGGCTGAAGC TGGGGTCTGG ACCTTCAATC AAAGCCTTAG 201ATGGGAGATC TCAAGTTTCA ACACCACGTT TTGGCAAAAC GTTCGATGCC 251 CCACCAGCCTTACCTAAAGC TACTAGAAAG GCTTTGGGAA CTGTCAACAG 301 AGCTACAGAA AAGTCTGTAAAGACCAAGGG ACCCCTCAAA CAAAAACAGC 351 CAAGCTTTTC TGCCAAAAAG ATGACTGAGAAGACTGTTAA AGCAAAAAGC 401 TCTGTTCCTG CCTCAGATGA TGCCTATCCA GAAATAGAAAAATTCTTTCC 451 CTTCAATCCT CTAGACTTTG AGAGTTTTGA CCTGCCTGAA GAGCACCAGA501 TTGCGCACCT CCCCTTGAGT GGAGTGCCTC TCATGATCCT TGACGAGGAG 551AGAGAGCTTG AAAAGCTGTT TCAGCTGGGC CCCCCTTCAC CTGTGAAGAT 601 GCCCTCTCCACCATGGGAAT CCAATCTGTT GCAGTCTCCT TCAAGCATTC 651 TGTCGACCCT GGATGTTGAATTGCCACCTG TTTGCTGTGA CATAGATATT 701 TAAATTTCTT AGTGCTTCAG AGTTTGTGTGTATTTGTATT AATAAAGCAT 751 TCTTTAACAG ATAAAAAAAA AAAAAAAAA (SEQ. ID.NO.:3).

The 3′ coding region of PTTG1 includes the following 168-nucleotidesequence, which corresponds to nucleotide positions 533 through 700 ofSEQ. ID. NO.:3, shown in Table 2 below.

TABLE 2 Portion of 3′ coding region of PTTG1 1 ATGATCCTTTG ACGAGGAGAGAGAGCTTGAA AAGCTGTTTC AGCTGGGCCC 51 CCCTTCACCT GTGAAGATGC CCTCTCCACCATGGGAATCC AATCTGTTGC 101 AGTCTCCTTC AAGCATTCTG TCGACCCTGG ATGTTGAATTGCCACCTGTT 151 TGCTGTGACA TAGATATT (SEQ.ID. NO.:10).

Another useful example of a PTTG-specific coding sequence is a sequencethat encodes a rat PTTG peptide, including nucleotide positions 293through 889 of SEQ. ID. NO.:1 (Table 3 below).

TABLE 3 Rat PTTG sequence. AATTCGGCAC GAGCCAACCT TGAGCATCTG ATCCTCTTGGCTTCTCCTTC CTATCGCTGA 60 (SEQ. ID. NO.:1) GCTGGTAGGC TGGAGACAGTTGTTTGGGTG CCAACATCAA CAAACGATTT CTGTAGTTTA 120 GCGTTTATGA CCCTGGCGTGAAGATTTAAG GTCTGGATTA AGCCTGTTGA CTTCTCCAGC 180 TACTTCTAAA TTTTTGTGCATAGGTGCTCT GGTCTCTGTT GCTGCTTAGT TCTTCCAGCC 240 TTCCTCAATG CCAGTTTTATAATATGCAGG TCTCTCCCCT CAGTAATCCA GG ATG 295 GCT ACT CTG ATC TTT GTT GATAAG GAT AAC GAA GAG CCA GGC AGC CGT 343 TTG GCA TCT AAG GAT GGA TTG AAGCTG GGC TCT GGT GTC AAA GCC TTA 391 GAT GGG AAA TTG CAG GTT TCA ACG CCACGA GTC GGC AAA GTG TTC GGT 439 GCC CCA GGC TTG CCT AAA GCC AGC AGG AAGGCT CTG GGA ACT GTC AAC 487 AGA GTT ACT GAA AAG CCA GTG AAG AGT AGT AAACCC CTG CAA TCG AAA 535 CAG CCG ACT CTG AGT GTG AAA AAG ATC ACC GAG AAGTCT ACT AAG ACA 583 CAA GGC TCT GCT CCT GCT CCT GAT GAT GCC TAC CCA GAAATA GAA AAG 631 TTC TTC CCC TTC GAT CCT CTA GAT TTT GAG AGT TTT GAC CTGCCT GAA 679 GAG CAC CAG ATC TCA CTT CTC CCC TTG AAT GGA GTG CCT CTC ATGATC 727 CTG AAT GAA GAG AGG GGG CTT GAG AAG CTG CTG CAC CTG GAC CCC CCT775 TCC CCT CTG CAG AAG CCC TTC CTA CCG TGG GAA TCT GAT CCG TTG CCG 823TCT CCT CCC AGC GCC CTC TCC GCT CTG GAT GTT GAA TTG CCG CCT GTT 871 TGTTAC GAT GCA GAT ATT TAAACGTCTT ACTCCTTTAT AGTTTATGTA 919 AGTTGTATTAATAAAGCATT TGTGTGTAAA AAAAAAAAAA AAAACTCGAG AGTAC 974The 3′ coding region of rat PTTG includes the following 168-nucleotidesequence, which corresponds to nucleotide positions 722 through 889 ofSEQ. ID. NO.:1, shown in Table 4 below.

TABLE 4 Portion of 3′ coding region of rat PTTG. ATG ATC CTG AAT GAA GAGAGG GGG CTT GAG AAG CTG CTG CAC CTG GAC 48 CCC CCT TCC CCT CTG CAG AAGCCC TTC CTA CCG TGG GAA TCT GAT CCG 96 TTG CCG TCT CCT CCC AGC GCC CTCTCC GCT CTG GAT GTT GAA TTG CCG 144 CCT GTT TGT TAC GAT GCA GAT ATT 168(SEQ. ID. NO.:18).

Another useful example of a PTTG-specific coding sequence is a sequencethat encodes a murine PTTG peptide, including nucleotide positions 304through 891 of SEQ. ID. NO.:15 (Table 5 below).

TABLE 5 Murine PTTG sequence. 1 TCTTGAACTT GTTATGTAGC AGGAGGCCAAATTTGAGCAT CCTCTTGGCT TCTCTTTATA 61 GCAGAGATTG TAGGCTGGAG ACAGTTTTGATGGGTGCCAA CATAAACTGA TTTCTGTAAG 121 AGTTGAGTGT TTTATGACCC TGGCGTGCAGATTTAGGATC TGGATTAAGC CTGTTGACTT 181 CTCCAGCTAC TTATAAATTT TTGTGCATAGGTGCCCTGGG TAAAGCTTGG TCTCTGTTAC 241 TGCGTAGTTT TTCCAGCCGT CTCAATGCCAATATTCAGGC TCTCTCCCTT AGAGTAATCC 301 AGAATGGCTA CTCTTATCTT TGTTGATAAGGATAATGAAG AACCCGGCCG CCGTTTGGCA 361 TCTAAGGATG GGTTGAAGCT GGGCACTGGTGTCAAGGCCT TAGATGGGAA ATTGCAGGTT 421 TCAACGCCTC GAGTCGGCAA AGTGTTCAATGCTCCAGCCG TGCCTAAAGC CAGCAGAAAG 481 GCTTTGGGGA CAGTCAACAG AGTTGCCGAAAAGCCTATGA AGACTGGCAA ACCCCTCCAA 541 CCAAAACAGC CGACCTTGAC TGGGAAAAAGATCACCGAGA AGTCTACTAA GACACAAAGC 601 TCTGTTCCTG CTCCTGATGA TGCCTACCCAGAAATAGAAA AGTTCTTCCC TTTCAATCCT 661 CTAGATTTTG ACCTGCCTGA GGAGCACCAGATCTCACTTC TCCCCTTGAA TGGCGTGCCT 721 CTCATCACCC TGAATGAAGA GAGAGGGCTGGAGAAGCTGC TGCATCTGGG CCCCCCTAGC 781 CCTCTGAAGA CACCCTTTCT ATCATGGGAATCTGATCCGC TGTACTCTCC TCCCAGTGCC 841 CTCTCCACTC TGGATGTTGA ATTGCCGCCTGTTTGTTACG ATGCAGATAT TTAAACTTCT 901 TACTTCTTTG TAGTTTCTGT ATGTATGTTGTATTAATAAA GCATT (SEQ. ID. NO.:15).

The 3′ coding region of murine PTTG includes the following168-nucleotide sequence, which corresponds to nucleotide positions 724through 891 of SEQ. ID. NO.:15, shown in Table 6 below.

TABLE 6 Portion of 3′ coding region of murine PTTG. ATCACCCTGAATGAAGAGAG AGGGCTGGAG AAGCTGCTGC ATCTGGGCCC CCCTAGCCCT 60 CTGAAGACACCCTTTCTATC ATGGGAATCT GATCCGCTGT ACTCTCCTCC CAGTGCCCTC 120 TCCACTCTGGATGTTGAATT GCCGCCTGTT TGTTACGATG CAGATATT 168 (SEQ. ID. NO.:19).

Inventive PTTG-C-related polynucleotides having nucleotides sequences ofSEQ. ID. NOS.:10, 18, or 19, degenerate coding sequences, or sequencescomplementary to any of these, are merely illustrative of useful PTTGcarboxy-terminal-related polynucleotides. Other useful PTTGcarboxy-terminal-related polynucleotides are functional fragments of anyof SEQ. ID. NOS.:10, 18, or 19 at least about 45 contiguous nucleotideslong, degenerate coding sequences, or sequences complementary to any ofthese, the presence of which in the cell can function to downregulateendogenous PTTG expression and/or PTTG function, which functionality canbe determined by routine screening.

As used herein, the term “degenerate” refers to codons that differ in atleast one nucleotide from a reference nucleic acid, e.g., SEQ ID NOS:1,3, 10, 15, 18, or 19, but encode the same amino acids as the referencenucleic acid. For example, codons specified by the triplets “UCU”,“UCC”, “UCA”, and “UCG” are degenerate with respect to each other sinceall four of these codons encode the amino acid serine.

Other useful polynucleotides include nucleic acids or otherpolynucleotides, that differ in sequence from the sequences shown in SEQID NO:1, SEQ. ID. NO.:3, SEQ. ID. NO.:10, SEQ. ID. NO.:15, SEQ. ID.NO.:18, and SEQ. ID. NO.:19, but which when expressed in a cell, resultin the same phenotype. Phenotypically similar nucleic acids are alsoreferred to as “functionally equivalent nucleic acids”. As used herein,the phrase “functionally equivalent nucleic acids” encompasses nucleicacids characterized by slight and non-consequential sequence variationsthat will function in substantially the same manner, compared to any ofthe detailed nucleotide sequences disclosed herein, to produce PTTGprotein functional with respect to inducing neoplastic cellularproliferation and/or transformation, or PTTG-C peptide(s) functionalwith respect to inhibition of neoplastic cellular proliferation and/ortransformation, and/or polypeptide products functional with respect toimmunogenicity. Such polynucleotides can have substantially the samecoding sequences as the reference sequences, encoding the amino acidsequence as set forth in SEQ. ID. NO.:2, SEQ. ID. NO.:4, SEQ. ID. NO.:9,SEQ. ID. NO:14, SEQ. ID. NO.:16, or SEQ. ID. NO.:17 or a larger aminoacid sequence including SEQ. ID. NO.:2, SEQ. ID. NO.:4, SEQ. ID. NO.:9,SEQ. ID. NO.:14, SEQ. ID. NO.:16, or SEQ. ID. NO.:17. As employedherein, the term “substantially the same nucleotide sequence” refers toDNA having sufficient identity to the reference polynucleotide, suchthat it will hybridize to the reference nucleotide under moderatelystringent hybridization conditions. In other embodiments, DNA having“substantially the same nucleotide sequence” as the reference nucleotidesequence has at least about 60% identity with respect to the referencenucleotide sequence. DNA having at least 70%, more preferably at least90%, yet more preferably at least 95%, identity to the referencenucleotide sequence is preferred.

In preferred embodiments, functionally equivalent nucleic acids encodepolypeptides or peptide fragments that are the same as those disclosedherein or that have conservative amino acid variations, or that encodelarger polypeptides that include SEQ. ID. NO.:2, SEQ. ID. NO.:4, SEQ.ID. NO.:9, or SEQ. ID. NO.:14, SEQ. ID. NO.:16, or SEQ. ID. NO.:17, orfragments of any of these that are biologically functional fragmentswith respect to inhibiting neoplastic cellular proliferation and/ortransformation. For example, conservative variations includesubstitution of a non-polar residue with another non-polar residue, orsubstitution of a charged residue with a similarly charged residue.These variations include those recognized by skilled artisans as thosethat do not substantially alter the tertiary structure of the protein.

Useful polynucleotides can be produced by a variety of methodswell-known in the art, e.g., by employing PCR and other similaramplification techniques, using oligonucleotide primers specific tovarious regions of SEQ ID NOS:1, 3, 10, 15, 18, 19, or functionallyequivalent polynucleotide sequences. Other synthetic methods forproducing polynucleotides or oligonucleotides of various lengths arealso well known.

In accordance with the method, preferred polynucleotides hybridize undermoderately stringent, preferably high stringency, conditions tosubstantially the entire sequence, or substantial portions (i.e.,typically at least 15-30 nucleotide) of the nucleic acid sequence setforth in SEQ ID NOS:1, 3, 10, 15, 18, or 19, or to complementarysequences.

The phrase “stringent hybridization” is used herein to refer toconditions under which annealed hybrids, or at least partially annealedhybrids, of polynucleic acids or other polynucleotides are stable. Asknown to those of skill in the art, the stability of hybrids isreflected in the melting temperature (T_(m)) of the hybrids. In general,the stability of a hybrid is a function of sodium ion concentration andtemperature. Typically, the hybridization reaction is performed underconditions of relatively low stringency, followed by washes of varying,but higher, stringency. Reference to hybridization stringency relates tosuch washing conditions.

As used herein, the phrase “moderately stringent hybridization” refersto conditions that permit target-DNA to bind a complementary nucleicacid that has about 60% sequence identity or homology, preferably about75% identity, more preferably about 85% identity to the target DNA; withgreater than about 90% identity to target-DNA being especiallypreferred. Preferably, moderately stringent conditions are conditionsequivalent to hybridization in 50% formamide, 5× Denhart's solution,5×SSPE, 0.2% SDS at 42° C., followed by washing in 0.2×SSPE, 0.2% SDS,at 65° C.

The phrase “high stringency hybridization” refers to conditions thatpermit hybridization of only those nucleic acid sequences that formstable hybrids in 0.018 M NaCl at 65° C. (i.e., if a hybrid is notstable in 0.018 M NaCl at 65° C., it will not be stable under highstringency conditions, as contemplated herein). High stringencyconditions can be provided, for example, by hybridization in 50%formamide, 5× Denhart's solution, 5×SSPE, 0.2% SDS at 42° C., followedby washing in 0.1×SSPE, and 0.1% SDS at 65° C.

The phrase “low stringency hybridization” refers to conditionsequivalent to hybridization in 10% formamide, 5× Denhart's solution,6×SSPE, 0.2% SDS at 42° C., followed by washing in 1×SSPE, 0.2% SDS, at50° C. Denhart's solution and SSPE (see, e.g., Sambrook et al.,Molecular Cloning, A Laboratory Manual, Cold Spring Harbor LaboratoryPress [1989]) are well known to those of skill in the art as are othersuitable hybridization buffers.

The PTTG carboxy-terminal-related polynucleotide can be, but is notnecessarily, of homologous origin with respect to the cell, due to therelatively high degree of sequence homology among mammalian PTTGsequences. PTTG carboxy-terminal-related polynucleotides of heterologousmammalian origin with respect to the cell is also useful. Thus, forexample, in accordance with the inventive method, a humanPTTG-C-encoding sequence functions to down regulate endogenous PTTGexpression and/or PTTG function in cells of non-human mammalian origin,such as murine or rat cells, and vice versa.

In preferred embodiments of the method of inhibiting neoplastic cellularproliferation and/or transformation of a mammalian cell, thepolynucleotide is complexed with a cellular uptake-enhancing agent, inan amount and under conditions sufficient to enter the cell. An“uptake-enhancing” agent, as utilized herein, means a composition ofmatter for enhancing the uptake of exogenous polynucleotides, such asDNA segment(s), nucleic acid analogs, or nucleic acid constructs, into aeukaryotic cell, preferably a mammalian cell, and more preferably ahuman cell. The enhancement is measured relative to the polynucleotideuptake in the absence of the uptake-enhancing agent, in the process oftransfecting or transducing the cell. Complexation with uptake-enhancingagent(s) generally augments the uptake of a polynucleotide into the celland/or reduces its breakdown by nucleases during its passage through thecytoplasm.

In accordance with preferred embodiments of the inventive method, PTTGcarboxy-terminal-related polynucleotides or PTTG-C peptides arecomplexed with an uptake-enhancing agent. “Complexed” means that thepolynucleotide or peptide is a constituent or member of a complex,mixture, or adduct resulting from chemical binding or bonding betweenand/or among the other constituents, including the cellularuptake-enhancing agent(s), and/or their moieties. Chemical binding orbonding can have the nature of a covalent bond, ionic bond, hydrogenbond, hydrophobic bond, or any combination of these bonding typeslinking the constituents of the complex at any of their parts ormoieties, of which a constituent can have one or a multiplicity ofmoieties of various sorts. Not every constituent of a complex need bebound to every other constituent, but each constituent has at least onechemical bond with at least one other constituent of the complex.Constituents can include, but are not limited to, molecular compounds ofa polar, non-polar, or detergent character; ions, including cations,such as, but not limited to, Na⁺, K⁺, Li⁺, Ca²⁺, Mg²⁺, Fe²⁺, Fe³⁺, Zn²⁺,Cu⁺, Cu²⁺, and/or NH₄ ⁺, or anions, such as, but not limited to Cl⁻,Br⁻, Fl⁻, NO₃ ⁻, NO₂ ⁻, NO⁻, HCO₃ ⁻, CO₃ ²⁻, SO₄ ²⁻, and/or PO₄ ³⁻;biological molecules, such as proteins, oligopeptides, polypeptides,oligonucleotides, nucleic acids, nucleic acid constructs, plasmids,viral particles; an/or organic polymers and co-polymers.

PTTG carboxy-terminal-related polynucleotides or PTTG-C peptides can be,but are not necessarily, directly bound to the cellular uptake-enhancingagent. For example, the polynucleotide can be contained in an expressionvector or other nucleic acid construct, which vector or other constructis bound to the uptake-enhancing agent at some moiety or part of hevector or construct not directly linked to the PTTGcarboxy-terminal-related polynucleotide; for purposes of the presentinvention, the PTTG carboxy-terminal-related polynucleotide is still“complexed” with the uptake-enhancing agent, although not being directlybound to the uptake-enhancing agent by a chemical bond. As long as thepolynucleotide and the uptake enhancing agent are both constituents ormembers of the same complex, an indirect chemical linkage suffices. Anexample with respect to PTTG-C peptides, is an intervening third peptidesequence linking a first PTTG-C peptide segment with a second celluptake-enhancing and/or importation-competent peptide segment. The firstand second peptide segments, indirectly linked, are “complexed” forpurposes of the invention.

Examples of uptake-enhancing agents usefully complexed with thepolynucleotide include cationic or polycationic lipid-DNA orliposome-DNA complexes (“lipoplexes”). Such lipoplexes can, optionally,also be coated with serum albumin or formulated as large-sizedcolloidally unstable complexes to further enhance transfectionefficiency; the presence of calcium di-cations (Ca²⁺) can also enhancelipid-based transfection efficiency. (E.g., Simoes, S. et al., Humanserum albumin enhances DNA transfection by lipoplexes and confersresistance to inhibition by serum, Biochim. Biophys. Acta 1463(2):459-69[2000]; Turek, J. et al., Formulations which increase the size oflipoplexes prevent serum-associated inhibition of transfection, J. GeneMed. 2(1):32-40 [2000]; Zudam, N. J. et al., Lamellarity of cationicliposomes and mode of preparation of lipoplexes affect transfectionefficiency, Biochim. Biophys. Acta 1419(2):207-20 [1999]; Lam, A. M. andCullis, P. R., Calcium enhances the transfection potency of plasmidDNA-cationic liposome complexes, Biochim. Biophys. Acta 1463(2):279-290[2000]).

Inventive compositions can include negatively charged ternary complexesof cationic liposomes, transferrin or fusigenic peptide(s)orpoly(ethylenimine). (E.g., Simoes, S. et al., Gene delivery bynegatively charged ternary complexes of DNA, cationic liposomes andtransferrin or fusigenic peptides, Gene Ther. 5(7):955-64 [1998]).Liposomal uptake-enhancing agents complexed with inventivepolynucleotide(s) can also be encapsulated in polyethylene glycol (PEG),FuGENE6, or the like. (E.g., Saravolac, E. G., et al., Encapsulation ofplasmid DNA in stabilized plasmid-lipid particles composed of differentcationic lipid concentration for optimal transfection activity, J. DrugTarget 7(6):423-37 [2000]; Yu, R. Z. et al., Pharmacokinetics and tissuedisposition in monkeys of an antisense oligonucleotide inhibitor ofHa-ras encapsulated in stealth liposomes, Pharm. Res. 16(8):1309-15[1999]; Tao, M. et al., Specific inhibition of human telomerase activityby transfection reagent, FuGENE6-antisense phophorothioateoligonucleotide complex in HeLa cells, FEBS Lett 454(3):312-6 [1999]).

In some embodiments, the uptake of antisense oligonucleotides is alsoenhanced by complexation with biocompatible polymeric or co-polymericnanoparticles, for example, comprising alginate, aminoalkylmethacrylate,methylmethacrylate, polymethylmethacrylate,methylaminoethyl-methacrylate, polyalkylcyanoacrylate (e.g.,polyhexylcyanoacrylate), or the like. (E.g., Aynie, I. et al.,Spongelike alginate nanoparticles as a new potential system for thedelivery of antisense oligonucleotides, Antisense Nucleic Acid Drug Dev.9(3):301-12 [1999]; Zimmer, A., Antisense oligonucleotide delivery withpolyhexylcyanoacrylate nanoparticles as carriers, Methods 18(3):286-95,322 [1999]; Berton, M. et al., Highly loaded nanoparticulate carrierusing an hydrophobic antisense oligonucleotide complex, Eur. J. Pharm.Sci. 9(2):163-70 [1999]; Zobel, H. P. et al., Evaluation ofaminoalkylmethacrylate nanoparticles as colloidal drug carrier systems.Part II: characterization of antisense oligonucleotides loaded copolymernanoparticles, Eur. J. Pharm. Biopharm. 48(1):1-12 [1999]; Fattal, E. etal., Biodegradable polyalkylcyanoacrylate nanoparticles for the deliveryof oligonucleotides, J. Controlled Release 53(1-3):137-43 [1998]).

Other useful uptake-enhancing agents for complexing with polynucleotidesinclude starburst polyamidoamine (PAMAM) dendrimers. (E.g., Yoo, H. etal., PAMAM dendrimers as delivery agents for antisense oligonucleotides,Pharm. Res. 16(12): 1799-804 [1999]; Bielinska, A. U. et al.,Application of membrane-based dendrimer/DNA complexes for solid phasetransfection in vitro and in vivo, Biomaterials 21(9):877-87 [2000];Bielinska, A. U. et al., DNA complexing with polyamidoamine dendrimers:implications for transfection, Bioconjug. Chem. 10(5):843-50 [1999];Bielinska, A. U. et al., Regulation of in vitro gene expression usingantisense oligonucleotides or antisense expression plasmids transfectedusing starburst PAMAM dendrimers, Nucleic Acid Res. 24(11):2176-82[1996]; Kukowska-Latallo, J. F. et al., Efficient transfer of geneticmaterial into mammalian cells using Starburst polyamidoamine dendrimers,Proc. Natl. Acad. Sci. USA 93(10):4897-902 [1996]; Delong, R. et al.,Characterization of complexes of oligonucleotides with polyamidoaminestarburst dendrimers and effects on intracellular delivery, J. Pharm.Sci. 86(6):762-64 [1997]).

Other preferred uptake-enhancing agents include lipofectin,lipfectamine, DIMRIE C, Superfect, Effectin (Qiagen), unifectin,maxifectin, DOTMA, DOGS (Transfectam; dioctadecylamidoglycylspermine),DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DOTAP(1,2-dioleoyl-3-trimethylammoniumpropane), DDAB (dimethyldioctadecylammonium bromide), DHDEAB(N,N-di-n-hexadecyl-N,N-dihydroxyethyl ammonium bromide), HDEAB(N-n-hexadecyl-N,N-dihydroxyethylammonium bromide), polybrene, orpoly(ethylenimine) (PEI), and/or peptides, such as polylysine,protamine, pK17, peptide K8, and peptide p2. (E.g., Ferkol, Jr. et. al.,U.S. Pat. Nos., 5,972,900 and 5,972,901; Vaysse, L. and Arveiler, B.,Transfection using synthetic peptides: comparison of threeDNA-compacting peptides and effect of centrifugation, Biochim. Biophys.Acta 1474(2):244-50 [2000]; Ni, Y. H. et al., Protamine enhance theefficiency of liposome-mediated gene transfer in a cultured humanhepatoma cell line, J. Formos. Med. Assoc. 98(8):562-66 [1999];Banerjee, R. et al., Novel series of non-glycerol-based cationictransfection lipids for use in liposomal gene delivery, J. Med. Chem.42(21):4292-99 [1999]; Godbey, W. T. et al., Improved packing ofpoly(ethylenimine/DNA complexes increases transfection efficiency, GeneTher. 6(8):1380-88 [1999]; Kichler, A et al., Influence of the DNAcomplexation medium on the transfection efficiency of lipospermine/DNAparticles, Gene Ther. 5(6):855-60 [1998]; Birchaa, J. C. et al.,Physico-chemical characterisation and transfection efficiency oflipid-based gene delivery complexes, Int. J. Pharm. 183(2):195-207[1999]). These non-viral cellular uptake-enhancing agents have theadvantage that they facilitate stable integration of xenogeneic DNAsequences into the vertebrate genome, without size restrictions commonlyassociated with virus-derived transfecting or transducing agents.

Another example, a viral cellular uptake-enhancing agent, is theadenovirus enhanced transferrin-polylysine-mediated gene delivery systemhas been described and patented by Curiel et al. (Curiel D. T., et al.,Adenovirus enhancement of transferrin-polylysine-mediated gene delivery,PNAS USA 88: 8850-8854 (1991). The delivery of DNA depends uponendocytosis mediated by the transferrin receptor (Wagner et al.,Transferrin-polycation conjugates as carriers for DNA uptake into cells,PNAS (USA) 87: 3410-3414 (1990). In addition this method relies on thecapacity of adenoviruses to disrupt cell vesicles, such as endosomes andrelease the contents entrapped therein. This system can enhance the genedelivery to mammalian cells by as much as 2,000 fold over other methods.

The amount of each component of the composition is chosen so that thegene modification, e.g., by transfection or transduction, of a mammaliancell is optimized. Such optimization requires no more than routineexperimentation. The ratio of polynucleotide to lipid is broad,preferably about 1:1, although other effective proportions can also beutilized depending on the type of lipid uptake-enhancing agent andpolynucleotide utilized. (E.g., Banerjee, R. et al. [1999];Jaaskelainen, I. et al., A lipid carrier with a membrane activecomponent and a small complex size are required for efficient cellulardelivery of anti-sense phosphorothioate oligonucleotides, Eur. J. Pharm.Sci. 10(3):187-193 [2000]; Sakurai, F. et al., Effect of DNA/liposomemixing ratio on the physicochemical characteristics, cellular uptake andintracellular trafficking of plasmid DNA/cationic liposome complexes andsubsequent gene expression, J. Controlled Release 66(2-3):255-69[2000]).

A suitable amount of the inventive polynucleotide to be delivered to thecells, in accordance with the method, preferably ranges from about 0.1nanograms to about 1 milligram per gram of tumor tissue, in vivo, orabout 0.1 nanograms to about 1 microgram per 5000 cells, in vitro.Suitable amounts for particular varieties of PTTG-C-relatedpolynucleotides and/or cell types and/or for various mammalian subjectsundergoing treatment, can be determined by routine experimentation. Forexample, malignant cell lines, such as MCF-7 or HeLa, typically are moreefficiently transfected by the inventive PTTG-C-related polynucleotidesthan non-malignant cell lines. Also, those skilled in the art are awarethat there is typically considerable variability among individual cancerpatients to any single treatment regimen, therefore, the practitionerwill tailor any embodiment of the inventive method to each individualpatient as appropriate.

In some preferred embodiments, the polynucleotide can be delivered intothe mammalian cell, either in vivo or in vitro using suitable expressionvectors well-known in the art (e.g., retroviral vectors, adenovirusvectors, and the like). In addition, to inhibit the in vivo expressionof PTTG, the introduction by expression vector of the antisense strandof a DNA encoding a PTTG-C peptide is contemplated.

Suitable expression vectors are well-known in the art, and includevectors capable of expressing DNA operatively linked to a regulatorysequence, such as a promoter region that is capable of regulatingexpression of such DNA. Thus, an expression vector refers to arecombinant DNA or RNA construct, such as a plasmid, a phage,recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the inserted DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome.

Exemplary, eukaryotic expression vectors, include the cloned bovinepapilloma virus genome, the cloned genomes of the murine retroviruses,and eukaryotic cassettes, such as the pSV-2 gpt system (described byMulligan and Berg, 1979, Nature Vol. 277:108-114) the Okayama-Bergcloning system (Mol. Cell Biol. Vol. 2:161-170, 1982), pGAL4, pCI (e.g.,pCI-neo), and the expression cloning vector described by GeneticsInstitute (Science Vol. 228:810-815, 1985), are available which providesubstantial assurance of at least some expression of the protein ofinterest in the transformed mammalian cell.

Particularly preferred are vectors which contain regulatory elementsthat can be linked to the inventive PTTG-encoding DNAs, such as aPTTG-C-encoding DNA segment, for transfection of mammalian cells.Examples are cytomegalovirus (CMV) promoter-based vectors such as pcDNAI(Invitrogen, San Diego, Calif.), MMTV promoter-based vectors such aspMAMNeo (Clontech, Palo Alto, Calif.) and pMSG (Pharmacia, Piscataway,N.J.), and SV40 promoter-based vectors such as pSVβ (Clontech, PaloAlto, Calif.).

In one embodiment of the present invention,adenovirus-transferrin/polylysine-DNA (TfAdp1-DNA) vector complexes(Wagner et al., 1992, PNAS, USA, 89:6099-6103; Curiel et al., 1992, Hum.Gene Therapy, 3:147-154; Gao et al., 1993, Hum. Gene Ther., 4:14-24) areemployed to transduce mammalian cells with heterologous PTTG-specificnucleic acid. Any of the plasmid expression vectors described herein maybe employed in a TfAdp1-DNA complex.

In addition, vectors may contain appropriate packaging signals thatenable the vector to be packaged by a number of viral virions, e.g.,retroviruses, herpes viruses, adenoviruses, resulting in the formationof a “viral vector.”

“Virus”, as used herein, means any virus, or transfecting fragmentthereof, which can facilitate the delivery of the polynucleotide intomammalian cells. Examples of viruses which are suitable for use hereinare adenoviruses, adeno-associated viruses, retroviruses such as humanimmune-deficiency virus, lentiviruses, mumps virus, and transfectingfragments of any of these viruses, and other viral DNA segments thatfacilitate the uptake of the desired DNA segment by, and release into,the cytoplasm of germ cells and mixtures thereof. A preferred viralvector is Moloney murine leukemia virus and the retrovirus vectorderived from Moloney virus calledvesicular-stomatitis-virus-glycoprotein (VSV-G)-Moloney murine leukemiavirus. A most preferred viral vector is a pseudotyped (VSV-G) lentiviralvector derived from the HIV virus. (Naldini et al. [1996]). Also, themumps virus is particularly suited because of its affinity for immaturesperm cells including spermatogonia. All of the above viruses mayrequire modification to render them non-pathogenic or less antigenic.Other known viral vector systems, however, are also useful within theconfines of the invention.

Viral based systems provide the advantage of being able to introducerelatively high levels of the heterologous nucleic acid into a varietyof cells. Suitable viral vectors for introducing inventive PTTG-specificpolynucleotides into mammalian cells (e.g., vascular tissue segments)are well known in the art. These viral vectors include, for example,Herpes simplex virus vectors (e.g., Geller et al., 1988, Science,241:1667-1669), Vaccinia virus vectors (e.g., Piccini et al., 1987,Meth. in Enzymology, 153:545-563; Cytomegalovirus vectors (Mocarski etal., in Viral Vectors, Y. Gluzman and S. H. Hughes, Eds., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1988, pp. 78-84), Moloneymurine leukemia virus vectors (Danos et al., 1980, PNAS, USA, 85:6469),adenovirus vectors (e.g., Logan et al., 1984, PNAS, USA, 81:3655-3659;Jones et al., 1979, Cell, 17:683-689; Berkner, 1988, Biotechniques,6:616-626; Cotten et al., 1992, PNAS, USA, 89:6094-6098; Graham et al.,1991, Meth. Mol. Biol., 7:109-127), adeno-associated virus vectors,retrovirus vectors (see, e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764),and the like. Especially preferred viral vectors are the adenovirus andretroviral vectors.

As used herein, “retroviral vector” refers to the well-known genetransfer plasmids that have an expression cassette encoding anheterologous gene residing between two retroviral LTRs. Retroviralvectors typically contain appropriate packaging signals that enable theretroviral vector, or RNA transcribed using the retroviral vector as atemplate, to be packaged into a viral virion in an appropriate packagingcell line (see, e.g., U.S. Pat. No. 4,650,764). Retroviral vectorsinclude lentiviral vectors, such as HIV-derived vectors.

Suitable retroviral vectors for use herein are described, for example,in U.S. Pat. No. 5,252,479, and in WIPO publications WO 92/07573, WO90/06997, WO 89/05345, WO 92/05266 and WO 92/14829, incorporated hereinby reference, which provide a description of methods for efficientlyintroducing nucleic acids into human cells using such retroviralvectors. Other retroviral vectors include, for example, the mousemammary tumor virus vectors (e.g., Shackleford et al., 1988, PNAS, USA,85:9655-9659), and the like.

A most preferred embodiment employs a pseudotyped retroviral vectorsystem, which was developed for gene therapy. (Naldini, L., et al., Invivo gene delivery and stable transduction of nondividing cells by alentiviral vector, Science 272: 263-267 [1996]), and which is used totransduce mammalian cells. This gene delivery system employs retroviralparticles generated by a three-plasmid expression system. In this systema packaging construct contains the human cytomegalovirus (hCMV)immediate early promoter, driving the expression of all viral proteins.The construct's design eliminates the cis-acting sequences crucial forviral packaging, reverse transcription and integration of thesetranscripts. The second plasmid encodes a heterologous envelope protein(env), namely the G glycoprotein of the vesicular stomatitis virus(VSV-G). The third plasmid, the transducing vector (pHR′), containscis-acting sequences of human immunodeficiency virus (HIV) required forpackaging, reverse transcription and integration, as well as uniquerestriction sites for cloning heterologous complementary DNAs (cDNAs).For example, a genetic selection marker, such as green fluorescentprotein (GFP), enhanced green fluorescent protein (EGFP), bluefluorescent protein, yellow fluorescent protein, β-galactosidase, and/ora gene encoding another preselected product is cloned downstream of thehCMV promoter in the HR′ vector, and is operatively linked so as to forma transcriptional unit. A VSV-G pseudotyped retroviral vector system iscapable of infecting a wide variety of cells including cells fromdifferent species and of integrating into the genome. Some retroviruses,i.e., lentiviruses, such as HIV, have the ability to infect non-dividingcells. Lentiviruses have a limited capacity for heterologous DNAsequences, the size limit for this vector being 7-7.5 kilobases (Verma,I. M. and Somia, N., Gene Therapy—promises, problems and prospects,Nature 389:239-242 [1997]). In vivo experiments with lentiviruses showthat expression does not shut off like other retroviral vectors and thatin vivo expression in brain, muscle, liver or pancreatic-islet cells, issustained at least for over six months—the longest time tested so far(Verma and Somia [1997]; Anderson, W F., Human Gene Therapy, Nature(Suppl). 392:25-30 [1998]).

“Gene delivery (or transfection) mixture”, in the context of thispatent, means a selected PTTG carboxy-terminal-related polynucleotide,whether in sense or anti-sense orientation, together with an appropriatevector mixed, for example, with an effective amount of uptake-enhancingagent as described above. (E.g., Clark et al., Polycations and cationiclipids enhance adenovirus transduction and transgene expression in tumorcells, Cancer Gene Ther. 6(5):437-46 [1999]). For example, theefficiency of adenoviral-, retroviral-, or lentiviral-mediatedtransduction is enhanced significantly by including a cationic lipid,such as polybrene during the infection.

In peptide-based embodiments of the inventive method of inhibitingneoplastic cellular proliferation and/or transformation, involvesdelivering an inventive composition comprising a PTTG carboxy-terminalpeptide, which is interchangeably designated herein “PTTG-C” or “PTTGC-terminal peptide”.

The terms “protein”, “peptide”, and “polypeptide” are usedinterchangeably herein. As used herein, the phrase “PTTG” refers toprotein member of a mammalian family of PTTG proteins, formerly alsoknown as “pituitary-tumor-specific-gene” (PTSG) proteins, that are ableto transform mammalian cells in tissue culture (e.g., NIH 3T3 and thelike).

In vivo, PTTG proteins are further characterized by having the abilityto induce tumor formation, for example, in nude mice (e.g., whentransfected into NIH 3T3 and the like). PTTG proteins include naturallyoccurring allelic variants thereof encoded by mRNA generated byalternative splicing of a primary transcript, and further includefragments thereof which retain at least one native biological activity.

The term “biologically active” or “functional”, when used herein as amodifier of inventive PTTG protein(s), peptide(s), or fragments thereof,refers to a polypeptide that exhibits at least one of the functionalcharacteristics attributed to PTTG. For example, one biological activityof PTTG is the ability to transform cells in vitro (e.g., NIH 3T3 andthe like). Yet another biological activity of PTTG is the ability toinduce neoplastic cellular proliferation (e.g., tumorigenesis) in nudemice (e.g., when transfected into NIH 3T3 cells and the like).

On the other hand, the inventive PTTG-C peptide, as distinct from thefull length native PTTG protein, has the biological activity ofinhibiting PTTG -mediated tumorigenesis in a dominant negative manner.“Dominant negative” is commonly used to describe a gene or protein whichhas a dominant effect similar to that described genetically, i.e. onecopy of the gene gives a mutant phenotypic effect, and a negative effectin that it prevents or has a negative impact on a biological processsuch as a signal transduction pathway. Thus, PTTG carboxy-terminalpeptides have the ability to downregulate intracellular PTTG expressionand/or endogenous PTTG function. The inventive method is not limited toany particular biochemical, genetic, and/or physiological mechanism(s)by which a PTTG-C peptide exerts its biological activity on PTTGexpression and/or PTTG function, and any or all such mechanism(s) cancontribute to the biological activity of PTTG-C, in accordance with theinvention.

Another biological activity of PTTG or PTTG-C peptides is the ability toact as an immunogen for the production of polyclonal and monoclonalantibodies that bind specifically to PTTG and/or PTTG-C. Thus, aninventive nucleic acid encoding PTTG or PTTG-C will encode a polypeptidespecifically recognized by an antibody that also specifically recognizesa PTTG protein as described herein. Such activity may be assayed by anymethod known to those of skill in the art. For example, atest-polypeptide encoded by a PTTG cDNA can be used to produceantibodies, which are then assayed for their ability to bind to theprotein. If the antibody binds to the test-polypeptide and the proteinwith substantially the same affinity, then the polypeptide possesses therequisite biological activity with respect to immunogenicity.

In the method of inhibiting neoplastic cellular proliferation and/ortransformation of a mammalian cell, whether in vitro or in vivo, usefulPTTG-C peptides encompass also any fragment of a larger PTTG-C molecule,which fragment retains PTTG-C biological activity with respect todownregulating endogenous PTTG expression and/or endogenous PTTGfunction. Useful PTTG-C peptides are preferably, but not exclusively,about 15 to about 60 contiguous amino acid residues long and compriseone or more proline-rich regions, which are peptide segments having aPXXP motif, where the Xs between the proline (P) residues represent anyamino acid residue, including proline. The proline-rich region(s) of thePTTG-C peptide is a potential SH3-binding site.

Most preferably, the PTTG-C peptide is derived from a human PTTG, alsodesignated hPTTG or PTTG1 protein. The native human PTTG1 protein is 202amino acids long, having the following amino acid sequence (Table 7below; encoded by nucleotide positions 95 through 700 of human PTTG1sequence SEQ. ID. NO.:3 and degenerate sequences).

TABLE 7 PTTG1 amino acid sequence. 1 MATLIYVDKE NGEPGTRVVA KDGLKLGSGPSIKALDGRSQ VSTPRFGKTF (SEQ. ID. NO.:4) 51 DAPPALPKAT RKALGTVNRATEKSVKTKGP LKQKQPSFSA KKMTEKTVKA 101 KSSVPASDDA YPEIKFFPF NPLDFESFDLPEEHQIAHLP LSGVPLMILD 151 EERELEKLFQ LGPPSPVKMP SPPWESNLLQ SPSSILSTLDVELPPVCCDI 201 DIThe human PTTG1 peptide is also encoded by any degenerate codingsequence encoding the amino acid sequence of SEQ. ID. NO.:4.

A preferred PTTG-C has the amino acid sequence corresponding to aminoacid residues 147 through 202 of SEQ. ID. NO.:4 (Table 8 below; encodedby nucleotide positions 533 through 700 of SEQ. ID. NO.:3 or 1-168 ofSEQ. ID. NO.:10 and degenerate sequences).

TABLE 8 Human PTTG-C amino acid sequence. MILDEERELE KLFQLGPPSPVKMPSPPWES NLLQSPSSIL STLDVELPPV CCDIDI56 (SEQ. ID. NO.9).

There are at least two proline-rich regions between amino acid residues163-173 of SEQ. ID. NO.:4, which correspond to amino acid residues 17through 27 of SEQ. ID. NO.:9, encoded by nucleotides 49 through 81 ofSEQ. ID. NO.:10 and degenerate sequences. Proline-rich regions are foundat amino acid residues 163-167 and 170-173 of SEQ. ID. NO.:4,corresponding to amino acid residues 17-20 and 24-27 of SEQ. ID. NO.:9.Other useful smaller peptide fragments of SEQ. ID. NO.:9 are tested byroutine means for their effectiveness in inhibiting neoplastic cellularproliferation and/or transformation of a cell.

Another example of a PTTG protein is a rat PTTG having the followingamino acid sequence (Table 9 below; encoded by nucleotide positions293-889 of SEQ. ID. NO.:1 and degenerate sequences).

TABLE 9 Rat PTTG amino acid sequence. Met Ala Thr Leu Ile Phe Val AspLys Asp Asn Glu Glu Pro Gly Ser 16 Arg Leu Ala Ser Lys Asp Gly Leu LysLeu Gly Ser Gly Val Lys Ala 32 Leu Asp Gly Lys Leu Gln Val Ser Thr ProArg Val Gly Lys Val Phe 48 Gly Ala Pro Gly Leu Pro Lys Ala Ser Arg LysAla Leu Gly Thr Val 64 Asn Arg Val Thr Glu Lys Pro Val Lys Ser Ser LysPro Leu Gln Ser 80 Lys Gln Pro Thr Leu Ser Val Lys Lys Ile Thr Glu LysSer Thr Lys 96 Thr Gln Gly Ser Ala Pro Ala Pro Asp Asp Ala Tyr Pro GluIle Glu 112 Lys Phe Phe Pro Phe Asp Pro Leu Asp Phe Glu Ser Phe Asp LeuPro 128 Glu Glu His Gln Ile Ser Leu Leu Pro Leu Asn Gly Val Pro Leu Met144 Ile Leu Asn Glu Glu Arg Gly Leu Glu Lys Leu Leu His Leu Asp Pro 160Pro Ser Pro Leu Gln Lys Pro Phe Leu Pro Trp Glu Ser Asp Pro Leu 176 ProSer Pro Pro Ser Ala Leu Ser Ala Leu Asp Val Glu Leu Pro Pro 192 Val CysTyr Asp Ala Asp Ile 199 (SEQ. ID. NO.:2).

A rat PTTG-C peptide includes amino acid residues 144 through 199 ofSEQ. ID. NO.:2, i.e., SEQ. ID. NO.:16 (Table 10 below; encoded bynucleotide positions 722 through 889 of SEQ. ID. NO.:1 or 1-168 of SEQ.ID. NO.:18 and degenerate sequences).

TABLE 10 Rat PTTG-C amino sequence. Met Ile Leu Asn Glu Glu Arg Gly LeuGlu Lys Leu Leu His Leu Asp 16 SEQ. ID. NO.:16 Pro Pro Ser Pro Leu GlnLys Pro Phe Leu Pro Trp Glu Ser Asp Pro 32 Leu Pro Ser Pro Pro Ser AlaLeu Ser Ala Leu Asp Val Glu Leu Pro 48 Pro Val Cys Tyr Asp Ala Asp Ile56

The amino acid sequence of SEQ. ID. NO.:16 includes proline-rich regionsat amino acid residues 17-20, 24-27, and 34-37 (corresponding to aminoacid residues 160-163, 167-170, and 177-180 of SEQ. ID. NO.:2).

Another example of a PTTG protein is a murine PTTG having the followingamino acid sequence (Table 11 below; encoded by nucleotide positions 304through 891 of SEQ. ID. NO.:15 and degenerate sequences).

TABLE 11 Murine PTTG amino acid sequence. 1 MATLIFVDKD NEEPGRRLASKDGLKLGTGV KALDGKLQVS TPRVGKVFNA //SEQ ID NO: 14 51 PAVPKASRKALGTVNRVAEK PMKTGKPLQP KQPTLTGKKI TEKSTKTQSS 101 VPAPDDAYPE IEKEFPFNPLDFDLPEEHQI SLLPLNGVPL ITLNEERGLE 151 KLLHLGPPSP LKTPFLSWES DPLYSPPSALSTLDVELPPV CYDADI

A murine PTTG-C peptide includes amino acid residues 141 through 196 ofSEQ. ID. NO.:14, i.e., SEQ. ID. NO.:17 (Table 12 below; encoded bynucleotide positions 724 through 891 of SEQ. ID. NO.:15 or 1-168 of SEQ.ID. NO.:19 and degenerate sequences).

TABLE 12 Murine PTTG-C amino acid sequence. ITLNEERGLE KLLHLGPPSPLKTPFLSWES DPLYSPPSAL STLDVELPPV CYDADI 56 (SEQ. ID. NO.: 17).

The amino acid sequence of SEQ. ID. NO.:17 includes a proline-richregion at amino acid residues 17-20 (corresponding to amino acidresidues 157-160 of SEQ. ID. NO.:14).

Preferred PTTG-C peptides include:

(A) peptides having an amino acid sequence of (SEQ. ID. NO.:9), (SEQ.ID. NO.:16), or (SEQ. ID. NO.:17); or

(B) mammalian PTTG-C peptides having at least about 60% sequencehomology with any of the sequences in (A); or

(C) peptide fragments of any of the sequences in (A) or (B) thatcomprise at least 15 contiguous amino acid residues and that function todownregulate endogenous PTTG expression and/or PTTG function. Mostpreferably, the fragment of (C) includes one or more proline-richregions.

Those of skill in the art will recognize that in other useful PTTG-Cpeptides numerous residues of any of the above-described PTTG or PTTG-Camino acid sequences can be substituted with other, chemically,sterically and/or electronically similar residues without substantiallyaltering PTTG or PTTG-C biological activity. In addition, largerpolypeptide sequences containing substantially the same coding sequencesas in SEQ ID NO:2, SEQ. ID. NO.:4, SEQ. ID. NO.:9, SEQ. ID. NO.:14, SEQ.ID. NO.:16, or SEQ. ID. NO.:17 (e.g., splice variants) are contemplated.

As employed herein, the term “substantially the same amino acidsequence” refers to amino acid sequences having at least about 60%sequence homology or identity with respect to any of the amino acidsequences described herein (“reference sequences”), and retainingcomparable functional and biological activity characteristic of theprotein defined by the reference sequences described, particularly withrespect to neoplastic cellular proliferation and/or transformation orits inhibition. More preferably, proteins having “substantially the sameamino acid sequence” will have at least about 80%, still more preferablyabout 90% amino acid identity with respect to a reference amino acidsequence; with greater than about 95% amino acid sequence identity beingespecially preferred. It is recognized, however, that polypeptidecontaining less than the described levels of sequence identity arisingas splice variants or that are modified by conservative amino acidsubstitutions are also encompassed within the scope of the presentinvention. The degree of sequence homology is determined by conductingan amino acid sequence similarity search of a protein data base, such asthe database of the National Center for Biotechnology Information(NCBI), using a computerized algorithm, such as PowerBLAST, QBLAST,PSI-BLAST, PHI-BLAST, gapped or ungapped BLAST, or the “Align” programthrough the Baylor College of Medicine server. (E.g., Altchul, S. F., etal., Gapped BLAST and PSI-BLAST: a new generation of protein databasesearch programs, Nucleic Acids Res. 25(17):3389-402 [1997]; Zhang, J., &Madden, T. L., Power BLAST: a new network BLAST application forinteractive or automated sequence analysis and annotation, Genome Res.7(6):649-56 [1997]; Madden, T. L., et al., Applications of network BLASTserver, Methods Enzymol. 266:131-41 [1996]; Altschul, S. F., et al.,Basic local alignment search tool, J. Mol. Biol. 215(3):403-10 [1990]).

Also encompassed by the terms PTTG protein or PTTG-C peptide,respectively, are biologically functional or active peptide analogsthereof The term peptide “analog” includes any polypeptide having anamino acid residue sequence substantially identical to a sequencespecifically shown herein in which one or more residues have beenconservatively substituted with a functionally similar residue and whichdisplays the ability to mimic the biological activity of PTTG or PTTG-C,respectively, particularly with respect to neoplastic cellularproliferation and/or transformation or its inhibition as describedherein above. Examples of conservative substitutions include thesubstitution of one non-polar (hydrophobic) residue such as isoleucine,valine, leucine or methionine for another, the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, between glycine and serine, thesubstitution of one basic residue such as lysine, arginine or histidinefor another, or the substitution of one acidic residue, such as asparticacid or glutamic acid for another.

The phrase “conservative substitution” also includes the use of achemically derivatized residue in place of a non-derivatized residue,provided that such polypeptide displays the requisite biologicalactivity.

“Chemical derivative” refers to a subject polypeptide having one or moreresidues chemically derivatized by reaction of a functional side group.Such derivatized molecules include, for example, those molecules inwhich free amino groups have been derivatized to form aminehydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Freecarboxyl groups may be derivatized to form salts, methyl and ethylesters or other types of esters or hydrazides. Free hydroxyl groups maybe derivatized to form O-acyl or O-alkyl derivatives. The imidazolenitrogen of histidine may be derivatized to form N-im-benzylhistidine.Also included as chemical derivatives are those peptides which containone or more naturally occurring amino acid derivatives of the twentystandard amino acids. For example, 4-hydroxyproline may be substitutedfor proline; 5-hydroxylysine may be substituted for lysine;3-methylhistidine may be substituted for histidine; homoserine may besubstituted for serine; and ornithine may be substituted for lysine. Theinventive polypeptide of the present invention also include anypolypeptide having one or more additions and/or deletions of residues,relative to the sequence of a polypeptide whose sequence is shownherein, so long as the requisite PTTG or PTTG-C biological activity ismaintained.

In accordance with peptide-based embodiments of the inventive method ofinhibiting neoplastic cellular proliferation and/or transformation, thecomposition comprising the PTTG-C peptide is delivered to the cell. Asuitable amount of the inventive PTTG-C peptide to be delivered to thecells, in accordance with the method, preferably ranges from about 0.1nanograms to about 1 milligram per gram of tumor tissue, in vivo, orabout 0.1 nanograms to about 1 microgram per 5000 cells, in vitro.Suitable amounts for particular varieties of PTTG-C peptide and/or celltypes and/or for various individual mammalian subjects undergoingtreatment, can be determined by routine experimentation.

Methods of delivering and importing peptides into target cells areknown. For example, the composition preferably, but not necessarily,comprises in addition to the PTTG-C peptide, a complex in which thePTTG-C peptide is complexed with a cellular uptake-enhancing agent. Forexample, the PTTG-C peptide can be covalently linked in a complex to acellular uptake-enhancing and/or importation-competent peptide segmentfor delivery of PTTG-C into the mammalian cell; in addition, a nuclearlocalization peptide can be included in the complex to direct the PTTG-Cto the nucleus. (E.g., Lin et al., Method for importing biologicallyactive molecules into cells, U.S. Pat. No. 6,043,339). An“importation-competent peptide,” as used herein, is a sequence of aminoacids generally of a length of about 10 to about 50 or more amino acidresidues, many (typically about 55-60%) residues of which arehydrophobic such that they have a hydrophobic, lipid-soluble portion.The hydrophobic portion is a common, major motif of a signal peptide,and it is often recognizable as a central part of the signal peptide ofa protein secreted from cells. A signal peptide is a peptide capable ofpenetrating through the cell membrane to allow the export of cellularproteins. Signal peptides useful in the present method are also“importation-competent,” i.e., capable of penetrating through the cellmembrane from outside the cell to the interior of the cell.

In a preferred embodiment, a PTTG-C peptide forms a first PTTG-C peptidesegment of a chimeric or fusion protein. The chimeric or fusion proteincomprises at least the first PTTG-C peptide segment and a secondcellular uptake-enhancing and/or importation-competent peptide segment.The second segment of the chimeric or fusion protein is a cellularuptake-enhancing and/or importation-competent peptide segment, such as asignal peptide, that allows the hybrid molecule to enter neoplasticcells that overexpress PTTG, whether in vitro or in vivo. The secondpeptide segment, such as the human immunodeficiency virus (HIV) TATprotein (Schwarze, S. R., et al., In vivo protein transduction: deliveryof a biologically active protein into the mouse, Science 285:1569-72[1999]), infiltrates the cells, and once within the cells, the PTTG-Cpeptide segment of the fusion protein becomes active within the cells toinhibit endogenous PTTG expression and/or PTTG function. Another exampleof a useful uptake-enhancing peptide segment is the signal peptide fromKaposi fibroblast growth factor (K-FGF). But any cellularuptake-enhancing and/or importation-competent peptide segment, capableof translocating across the cell membrane into the interior of theselected target mammalian cell, can be used according to this invention.The chimeric or fusion protein can also include additional segments,such as a linker segment, that can be an intervening segment between thefirst and second segments. The additional segment can alternatively be aterminal segment, as appropriate.

In embodiments of the method involving the use of PTTG-C chimeric orfusion proteins, the cellular uptake-enhancing and/orimportation-competent peptide segment can be the uptake-enhancing agent.Alternatively, or in addition the cellular uptake-enhancing agent can bea lipid or liposome uptake-enhancing agent as described herein above,such as lipofectin, lipofectamine, DOTAP, and others. Cationic (orpolycationic) lipids or liposomes can also be complexed with a signalpeptide and a negatively-charged biologically active molecule by mixingthese components and allowing them to charge-associate. Anionicliposomes generally are utilized to encapsulate within the liposome thesubstances to be delivered to the cell. Procedures for forming cationicliposome-encapsulating substances are standard in the art and canreadily be utilized herein by one of ordinary skill in the art toencapsulate the complex of this invention. For example, liposomeuptake-enhancing agents complexed with inventive PTTG-C peptidefragments can be encapsulated in polyethylene glycol (PEG), FuGENE6, orthe like.

With respect to delivery of the composition to mammalian cells in vivo,the composition is administered to a mammalian subject in need oftreatment, including a human subject, by any conventional deliveryroute. Preferably, the PTTG-C peptide, whether or not complexed withcellular uptake-enhancing and/or importation-competent peptides (e.g.,signal or localization peptides), is injected intravenously,intra-arterially, intraperitoneally, or by means of injection directlyinto a tumor or into a cell by microinjection. Conventional stereotacticmethods can be useful for direct injection into tumors or cells. Inother preferred embodiments, controlled release formulations ofbiodegradable polymeric microspheres or nanospheres (e.g.,polylactide-co-glycolide; PLGA) encapsulating the PTTG-C peptide, orPTTG-C chimeric or fusion protein are administered to the mammaliansubject orally. (E.g., Zhu, G. et al., Stabilization of proteinsencapsulated in injectable poly(lactide-co-glycolide), NatureBiotechnology 18:52-57 [2000]). Administration by nasal, rectal, orvaginal delivery routes can also be useful. Administration by catheteror stent can also be useful for delivering the PTTG-C peptide.

In some embodiments, isolated and crystallized PTTG-C peptide can becross-linked with a multifunctional crosslinking agent that inhibitsproteolysis of the PTTG-C peptide in vivo. (Navia, M. A., Method ofprotein therapy by orally administering crosslinked protein crystals,U.S. Pat. No. 6,011,001).

In accordance with the inventive method of inhibiting neoplasticcellular proliferation and/or transformation that is mediated by PTTG,the mammalian cell is a cell that overexpresses PTTG, the gene thatencodes a PTTG protein. Although detecting PTTG overexpression by thecell is not essential or necessary to the practice of the inventivemethod, the level of PTTG expression, including overexpression, isdetectable by one skilled in the art. Detection of PTTG expression isaccomplished by immunochemical assay for PTTG protein, for example,using the inventive anti-PTTG-C antibodies, described herein, or otheranti-PTTG-specific antibodies. Alternatively, amplification ofPTTG-specific mRNAs present in biological samples (e.g., tissue biopsy)can be used to detect PTTG expression. This is done by known molecularbiological techniques of amplification and analysis of the amplificationproducts for the presence or absence of PTTG-specific amplificationproducts. If PTTG gene-specific amplification products are present, thefindings are indicative of expression of the PTTG gene and diagnostic ofthe presence of neoplastic cellular proliferation in the subject asdefined herein.

However, for interpretation of negatives (no PTTG-specific amplificationproducts) analysis is preferably carried out following a controlamplification of nucleic acids specific for a housekeeping gene, forexample, a gene encoding β-actin, phosphofructokinase (PFK),glyceraldehyde 3-phosphate dehydrogenase, or phosphoglycerate kinase.Only if expression of the housekeeping gene is detected in the sample,is the absence of PTTG gene expression reliably accepted. Withincreasing sensitivity of amplification and analysis methods employed,it becomes increasingly preferable to determine the level of PTTG geneexpression relative to expression of a housekeeping gene, in order tobetter distinguish neoplastic, hyperplastic, cytologically dysplasticand/or premalignant cellular growth or proliferation from the detectablebackground of normal cellular division. The ratio of PTTG expression tohousekeeping gene expression is determined, for example, by real-timePCR methods or densitometric measurement and analysis of electrophoreticbands after amplification. When the ratio of PTTG expression tohousekeeping gene expression exceeds a normal cell standard range and/orapproximates an abnormal (e.g., neoplastic) cell standard range, thisindicates overexpression of PTTG gene product, characteristic ofneoplastic, hyperplastic, cytologically dysplastic and/or premalignantcellular growth or proliferation.

PTTG-specific mRNAs in a biological sample are amplified by a suitableamplification method. For example, a reverse transcriptase-mediatedpolymerase chain reaction (RT-PCR) is employed to amplify PTTG-specificnucleic acids. Briefly, two enzymes are used in the amplificationprocess, a reverse transcriptase to transcribe PTTG-specific cDNA from aPTTG-specific mRNA template in the sample, a thermal resistant DNApolymerase (e.g., Taq polymerase), and PTTG-specific primers to amplifythe cDNA to produce PTTG gene-specific amplification products. The useof limited cycle PCR yields semi-quantitative results. (E.g., Gelfand etal., Reverse transcription with thermostable DNA polymerase-hightemperature reverse transcription, U.S. Pat. Nos. 5,310,652; 5,322,770;Gelfand et al., Unconventional nucleotide substitution in temperatureselective RT-PCR, U.S. Pat. No. 5,618,703).

Alternatively, single enzyme RT-PCR is employed to amplify PTTGgene-specific nucleic acids. Single enzymes now exist to perform bothreverse transcription and polymerase functions, in a single reaction.For example, the Perkin Elmer recombinant Thermus thermophilus (rTth)enzyme(Roche Molecular), or other similar enzymes, are commerciallyavailable.

Real-time RT-PCR can be employed to amplify PTTG-specific nucleic acids.Briefly, this is a quantitative gene analysis based on the ratio of PTTGgene expression and the expression of a housekeeping gene, i.e., a genethat is expressed at about the same level in normal and abnormal (e.g.,malignant) cells, for example, a gene encoding P-actin,phosphofructokinase, glyceraldehyde 3-phosphate dehydrogenase, orphosphoglyceratekinase. The the ratio of the PTTG and housekeepinggenes' expressions is routinely established as a standard for normal andabnormal cells, which standard expression ratio(s) is (are) used forcomparison in determining that expression of the PTTG gene relative toexpression of the “housekeeping” gene in a given sample is either“normal” or “increased”, the latter indicative of “overexpression” anddiagnostic for the presence of neoplastic, hyperplastic, cytologicallydysplastic and/or premalignant cellular growth or proliferation. In thisembodiment, the ratio is the key to diagnosis and constitutesquantitative gene expression analysis. This embodiment utilizesso-called real-time quantitative PCR, carried out with commerciallyavailable instruments, such as the Perkin Elmer ABI Prism 7700, theso-called Light Cycler (Roche Molecular), and/or other similarinstruments. Optionally, single enzyme RT-PCR technology, for example,employing rTth enzyme, can be used in a real-time PCR system.Preferably, amplification and analysis are carried out in an automatedfashion, with automated extraction of mRNA from a urine sediment sample,followed by real-time PCR, and fluorescence detection of amplificationproducts using probes, such as TaqMan or Molecular Beacon probes.Typically, the instrumentation includes software that providesquantitative analytical results during or directly following PCR withoutfurther amplification or analytical steps.

Alternatively, transcription-mediated amplification (TMA) is employed toamplify PTTG gene-specific nucleic acids. (E.g., K. Kamisango et al.,Quantitative detection of hepatitis B virus by transcription-mediatedamplification and hybridization protection assay, J. Clin. Microbiol.37(2):310-14 [1999]; M. Hirose et al., New method to measure telomeraseactivity by transcription-mediated amplification and hybridizationprotection assay, Clin. Chem. 44(12)2446-52 [1998]). Rather thanemploying RT-PCR for the amplification of a cDNA, TMA uses a probe thatrecognizes a PTTG-specific (target sequence) RNA; in subsequent steps,from a promoter sequence built into the probe, an RNA polymeraserepetitively transcribes a cDNA intermediate, in effect amplifying theoriginal RNA transcripts and any new copies created, for a level ofsensitivity approaching that of RT-PCR. The reaction takes placeisothermally (one temperature), rather than cycling through differenttemperatures as in PCR.

Other useful amplification methods include a reversetranscriptase-mediated ligase chain reaction (RT-LCR), which has utilitysimilar to RT-PCR. RT-LCR relies on reverse transcriptase to generatecDNA from mRNA, then DNA ligase to join adjacent syntheticoligonucleotides after they have bound the target cDNA.

Amplification of a PTTG gene-specific nucleic acid segment of thesubject can be achieved using PTTG gene-specific oligonucleotide primersand primer sets as provided herein.

Optionally, high throughput analysis may be achieved by PCR multiplexingtechniques well known in the art, employing multiple primer sets, forexample primers directed not only to PTTG gene-specific nucleic acids,but to amplifying expression products of housekeeping genes (controls)or of other potential diagnostic markers (e.g., oncogenes), as well,such as MAG or telomerase, to yield additional diagnostic information.(E.g., Z. Lin et al., Multiplex genotype determination at a large numberof gene loci, Proc. Natl. Acad. Sci. USA 93(6): 2582-87 [1996];Demetriou et al., Method and probe for detection of gene associated withliver neoplastic disease, U.S. Pat. No. 5,866,329).

Hybridization analysis is a preferred method of analyzing theamplification products, employing one or more PTTG-specific probe(s)that, under suitable conditions of stringency, hybridize(s) with singlestranded PTTG-specific nucleic acid amplification products comprisingcomplementary nucleotide sequences. Hybridization refers to the bindingof complementary strands of nucleic acid (i.e., sense:antisense strandsor probe:target-DNA) to each other through hydrogen bonds, similar tothe bonds that naturally occur in chromosomal DNA. The amplificationproducts are typically deposited on a substrate, such as a cellulose ornitrocellulose membrane, and then hybridized with labeled PTTG-specificprobe(s), optionally after an electrophoresis. Conventional dot blot,Southern, Northern, or fluorescence in situ (FISH) hybridizationprotocols, in liquid hybridization, hybridization protection assays, orother semi-quantitative or quantitative hybridization analysis methodsare usefully employed along with the PTTG gene-specific probes of thepresent invention. Preferred probe-based hybridization conditionscomprise a temperature of about 37° C., a formamide concentration ofabout 20%, and a salt concentration of about 5× standard saline citrate(SSC; 20×SSC contains 3 M sodium chloride, 0.3 M sodium citrate, pH7.0). Such conditions will allow the identification of sequences whichhave a substantial degree of similarity with the probe sequence, withoutrequiring perfect homology. The phrase “substantial similarity” refersto sequences which share at least 50% homology. Stringency levels usedto hybridize a given probe with target-DNA can be readily varied bythose of skill in the art. Preferably, hybridization conditions will beselected which allow the identification of sequences having at leastabout 60% homology with the probe, while discriminating againstsequences which have a lower degree of homology with the probe.

As used herein, a “probe” is single-stranded DNA or RNA, or a nucleicacid analog. The inventive probe is preferably 7 to 500 nucleotideslong, more preferably 14 to 150 nucleotides long, and most preferably atleast 50 nucleotides long. The probe comprises, for at least part of itslength, a PTTG-specific nucleotide sequence at least 7 to 15 contiguousnucleotides long, such that the probe hybridizes to a PTTG-specificsingle stranded nucleic acid under suitably stringent hybridizationconditions. Examples of PTTG-specific nucleotide sequences are set forthin any of SEQ. ID. NOS.: 1, 3, 10, 15, 18, or 19, preferably, but notnecessarily, including 5′ and/or 3′ coding regions thereof In addition,the entire cDNA encoding region of an inventive PTTG-specific nucleotidesequence, or the entire sequence corresponding to SEQ. ID. NOS.: 1, 3,10, 15, 18, 19, or nucleotide sequences complementary to any of these,can be used as a probe. For example, probes comprising inventiveoligonucleotide primer sequences, such as, but not limited to, SEQ. ID.NO.:8, can be labeled for use as probes for detecting or analyzingPTTG-specific nucleic acid amplification products. Any of the inventiveisolated PTTG-C-related polynucleotides can be used as probes orprimers.

Alternatively, electrophoresis for analyzing amplification products isdone rapidly and with high sensitivity by using any of various methodsof conventional slab or capillary electrophoresis, with which thepractitioner can optionally choose to employ any facilitating means ofnucleic acid fragment detection, including, but not limited to,radionuclides, UV-absorbance or laser-induced fluorescence. (K. Keparniket al., Fast detection of a (CA)18 microsatellite repeat in the IgEreceptor gene by capillary electrophoresis with laser-inducedfluorescence detection, Electrophoresis 19(2);249-55 [1998]; H. Inoue etal., Enhanced separation of DNA sequencing products by capillaryelectrophoresis using a stepwise gradient of electric field strength, J.Chromatogr. A. 802(1):179-84 [1998]; N. J. Dovichi, DNA sequencing bycapillary electrophoresis, Electrophoresis 18(12-13):2393-99 [1997]; H.Arakawa et al., Analysis of single-strand conformation polymorphisms bycapillary electrophoresis with laser induced fluorescence detection, J.Pharm. Biomed. Anal. 15(9-10):1537-44 [1997]; Y. Baba, Analysis ofdisease-causing genes and DNA-based drugs by capillary electrophoresis.Towards DNA diagnosis and gene therapy for human diseases, J. ChromatgrB. Biomed. Appl. 687(2):271-302 [1996]; K. C. Chan et al., High-speedelectrophoretic separation of DNA fragments using a short capillary, J.Chromatogr B. Biomed. Sci. Appl. 695(1):13-15 [1997]). Probes can belabeled by methods well-known in the art.

As used herein, the terms “label”, “tracer”, and “indicating means” intheir various grammatical forms refer to single atoms and molecules thatare either directly or indirectly involved in the production of adetectable signal. Any label or indicating means can be linked toPTTG-specific probes, primers, or amplification products, or PTTGproteins, peptides, peptide fragments, or anti-PTTG antibody molecules.The label can be used alone or in conjunction with additional reagents.Such labels are themselves well-known in the art. The label can be afluorescent labeling agent that chemically binds to antibodies orantigens without denaturation to form a fluorochrome (dye) that is auseful immunofluorescent tracer. A description of immunofluorescentanalytic techniques is found in DeLuca, “Immunofluorescence Analysis”,in Antibody As a Tool, Marchalonis et al., eds., John Wiley & Sons,Ltd., pp. 189-231 (1982), which is incorporated herein by reference. Anyof diverse fluorescent dyes can optionally be used as a label, includingbut not limited to, SYBR Green I Y1O-PRO-1, thiazole orange, Hex (i.e.,6-carboxy-2′,4′,7′,4,7-hexachlorofluoroscein), pico green, edans,fluorescein, FAM (i.e., 6-carboxyfluorescein), or TET (i.e.,4,7,2′,7′-tetrachloro-6-carboxyfluoroscein). (E.g., J. Skeidsvoll andP.M. Ueland, Analysis of double-stranded DNA by capillaryelectrophoresis with laser-induced fluorescence detection using themonomeric dye SYBR green I, Anal. Biochem. 231(20):359-65 [1995]; H.Iwahana et al., Multiple fluorescence-based PCR-SSCP analysis usinginternal fluorescent labeling of PCR products, Biotechniques21(30:510-14, 516-19 [1996]).

The label can also be an enzyme, such as horseradish peroxidase (HRP),glucose oxidase, β-galactosidase, and the like. Alternatively,radionuclides are employed as labels. The linking of a label to asubstrate, i.e., labeling of nucleic acid probes, antibodies,polypeptide, and proteins, is well known in the art. For instance, aninvention antibody can be labeled by metabolic incorporation ofradiolabeled amino acids provided in the culture medium. See, forexample, Galfre et al., Meth. Enzymol., 73:3-46 (1981). Conventionalmeans of protein conjugation or coupling by activated functional groupsare particularly applicable. See, for example, Aurameas et al., Scand.J. Immunol., Vol. 8, Suppl. 7:7-23 (1978), Rodwell et al., Biotech.,3:889-894 (1984), and U.S. Pat. No. 4,493,795.

In accordance with yet another embodiment of the present invention,there are provided anti-PTTG antibodies having specific reactivity withPTTG polypeptides of the present invention. Antibody fragments, forexample Fab, Fab′, F(ab′)₂, or F(v) fragments, that selectively orspecifically bind a PTTG protein, PTTG-C peptide, or immunogenicfragment of PTTG-C, are also encompassed within the definition of“antibody”.

Inventive antibodies can be produced by methods known in the art usingPTTG polypeptide, proteins or portions thereof, such as PTTG-C peptideor immunogenic fragments of PTTG-C, as antigens. For example, polyclonaland monoclonal antibodies can be produced by methods well known in theart, as described, for example, in Harlow and Lane, Antibodies: ALaboratory Manual (Cold Spring Harbor Laboratory [1988]), which isincorporated herein by reference. Isolated or purified PTTG proteins,PTTG-C peptides, and immunogenic PTTG-C fragments can be used asimmunogens in generating such specific antibodies.

PTTG proteins, PTTG-C peptides, or polypeptide analogs thereof, arepurified or isolated by a variety of known biochemical means, including,for example, by the recombinant expression systems described herein,precipitation, gel filtration, ion-exchange, reverse-phase and affinitychromatography, and the like. Other well-known methods are described inDeutscher et al., Guide to Protein Purification: Methods in EnzymologyVol. 182, (Academic Press, [1990]), which is incorporated herein byreference. Isolated PTTG proteins or PTTG-C peptides are free ofcellular components and/or contaminants normally associated with anative in vivo environment.

Isolated PTTG proteins or PTTG-C peptides can also be chemicallysynthesized For example, synthetic polypeptide can be produced usingApplied Biosystems, Inc. Model 430A or 431A automatic peptidesynthesizer (Foster City, Calif.) employing the chemistry provided bythe manufacturer. Alternatively, PTTG can be isolated or purified fromnative sources, and PTTG-C peptides can be isolated from PTTG (or fromchimeric proteins) by the use of suitable proteases.

Alternatively, PTTG or PTTG-C polypeptides can be recombinantly derived,for example, produced by mammalian cells genetically modified to expressPTTG-C-encoding polynucleotides in accordance with the inventivetechnology as described herein. Recombinant methods are well known, asdescribed, for example, in Sambrook et al., supra., 1989). An example ofthe means for preparing the inventive PTTG or PTTG-C polypeptide(s) isto express nucleic acids encoding the PTTG protein or PTTG-C peptide ina suitable host cell, such as a bacterial cell, a yeast cell, anamphibian cell (i.e., oocyte), or a mammalian cell, such as theinventive mammalian host cell described herein below, using methods wellknown in the art, and recovering the expressed polypeptide, again usingwell-known methods.

The immunogenicity of various PTTG-C fragments of interest is determinedby routine screening. Alternatively, synthetic PTTG or PTTG-Cpolypeptides or fragments thereof can be prepared (using commerciallyavailable synthesizers) and used as immunogens. Amino acid sequences canbe analyzed by methods well known in the art to determine whether theyencode hydrophobic or hydrophilic domains of the correspondingpolypeptide. Altered antibodies such as chimeric, humanized, CDR-graftedor bifunctional antibodies can also be produced by methods well known inthe art. Such antibodies can also be produced by hybridoma, chemicalsynthesis or recombinant methods described, for example, in Sambrook etal., supra., and Harlow and Lane, supra. Both anti-peptide andanti-fusion protein antibodies can be used. (see, for example, Bahouthet al., Trends Pharmacol. Sci. 12:338 [1991]; Ausubel et al., CurrentProtocols in Molecular Biology (John Wiley and Sons, NY [1989] which areincorporated herein by reference).

Antibody so produced can be used, inter alia, in diagnostic or assaymethods and systems to detect the level of PTTG protein, PTTG-C peptide,or immunogenic fragments thereof, present in a mammalian, preferablyhuman, biological sample, such as tissue or vascular fluid. This isuseful, for example, in determining the level of PTTG expression. Suchantibodies can also be used for the immunoaffinity or affinitychromatography purification of the inventive PTTG proteins or PTTG-Cpeptides. In addition, methods are contemplated herein for detecting thepresence of PTTG protein or PTTG-C peptide, either on the surface of acell or within a cell (such as within the nucleus), which methodscomprise contacting the cell with an antibody that specifically binds toPTTG protein or PTTG-C peptide, under conditions permitting specificbinding of the antibody to PTTG protein or PTTG-C peptide, detecting thepresence of the antibody bound to PTTG or PTTG-C, and thereby detectingthe presence of PTTG or PTTG-C polypeptide on the surface of, or within,the cell. With respect to the detection of such polypeptide, theantibodies can be used for in vitro diagnostic or assay methods, or invivo imaging methods.

Immunological procedures useful for in vitro detection of target PTTG orPTTG-C polypeptides in a sample include immunoassays that employ adetectable antibody. Such immunoassays include, for example, ELISA,immunofluorescence assay (IFA), Pandex microfluorimetric assay,agglutination assays, flow cytometry, serum diagnostic assays andimmunohistochemical staining procedures which are well known in the art.An antibody can be made detectable by various means well known in theart. For example, a detectable marker can be directly or indirectlyattached to the antibody. Useful markers include, for example,radionuclides, enzymes, fluorogens, chromogens and chemiluminescentlabels.

Inventive anti-PTTG or anti-PTTG-C antibodies are also contemplated foruse herein to modulate activity of the PTTG polypeptide in livinganimals, in humans, or in biological tissues or fluids isolatedtherefrom. Accordingly, compositions comprising a carrier and an amountof an antibody having specificity for PTTG polypeptide effective toblock naturally occurring ligands or other PTTG-binding proteins frombinding to invention PTTG polypeptide are contemplated herein. Forexample, a monoclonal antibody directed to an epitope of PTTGpolypeptide molecules present on the surface of a cell and having anamino acid sequence substantially the same as an amino acid sequence fora cell surface epitope of a PTTG polypeptide including the amino acidsequence shown in SEQ ID NOS:2, 4, 9, 14, 16, or 17 can be useful forthis purpose.

The present invention also relates to transfected, transduced, orotherwise transformed mammalian host cells comprising any of theinventive PTTG-C-related polynucleotide-containing compositions asdescribed herein above. The inventive cells are either contained in amammalian subject or are cultured in vitro. Included among preferredembodiments are mammalian host cells containing an expression vectorcomprising the inventive PTTG-C-related polynucleotide in atranscriptional unit. Preferably, a product is expressed by the cell,which product, most preferably, but not necessarily, is a biologicallyactive PTTG-C peptide that functions to downregulate PTTG-mediatedneoplastic cellular proliferation and/or transformation. In vitro and invivo methods of transfecting, transducing, or transforming suitable hostcells are generally known in the art. Methods for culturing cells, invitro, are also well known. Exemplary methods of transfection,transduction, or transformation include, e.g., infection employing viralvectors (see, e.g., U.S. Pat. Nos. 4,405,712 and 4,650,764), calciumphosphate transfection (U.S. Pat. Nos. 4,399,216 and 4,634,665), dextransulfate transfection, electroporation, lipofection (see, e.g., U.S. Pat.Nos. 4,394,448 and 4,619,794), cytofection, microparticle bombardment,and the like. The heterologous nucleic acid can optionally includesequences which allow for its extrachromosomal (i.e., episomal)maintenance, or heterologous DNA can be caused to integrate into thegenome of the host (as an alternative means to ensure stable maintenancein the host cell).

The present invention further provides transgenic non-human mammalscontaining the inventive mammalian cells that are capable of expressingexogenous nucleic acids encoding PTTG polypeptides, particularly theinventive PTTG-C peptides and functional fragments thereof as describedhereinabove. As employed herein, the phrase “exogenous nucleic acid”refers to nucleic acid sequence which is not native to the host, orwhich is present in the host in other than its native environment (e.g.,as part of a genetically engineered DNA construct). Methods of producingtransgenic non-human mammals are known in the art. Typically, thepronuclei of fertilized eggs are microinjected in vitro with foreign,i.e., xenogeneic or allogeneic DNA or hybrid DNA molecules, and themicroinjected fertilized eggs are then transferred to the genital tractof a pseudopregnant female to gestate to term. (E.g., P. J. A.Krimpenfort et al., Transgenic mice depleted in mature T-cells andmethods for making transgenic mice, U.S. Pat. Nos. 5,175,384 and5,434,340; P. J. A. Krimpenfort et al., Transgenic mice depleted inmature lymphocytic cell-type, U.S. Pat. No. 5,591,669). Alternatively,methods for producing transgenic non-human mammals can involve geneticmodification of female or male germ cells using an expression vector,which germ cells are then used to produce zygotes, which are gestated toterm. The resulting offspring are selected for the desired phenotype.These offspring can further be bred or cloned to produce additonalgenerations of transgenic animals with the desired phenotype. Theinventive transgenic non-human mammals, preferably, but not necessarily,are large animals such as bovines, ovines, porcines, equines, and thelike, that produce relatively large quantities of PTTG-C peptides thatcan be harvested for use in practicing the method of inhibitingneoplastic cellular proliferation and/or transformation.

Most preferably, the transgenic non-human mammal is a female thatproduces milk into which the inventive PTTG-C peptides have beensecreted. The PTTG-C peptides are then purified from the milk. (E.g.,Christa, L., et al., High expression of the humanhepatocarcinoma-intestine-pancreas/pancreatic-associated protein(HIPPAP) gene in the mammary gland of lactating transgenic micesecretion into the milk and purification of the HIP/PAP lectin, Eur. J.Biochem. 267(6):1665-71 [2000]; Sobolev, A. S. et al., Receptor-mediatedtransfection of murine and ovine mammary glands in vivo, J. Biol. Chem.273(14):7928-33 [1998]; Zhang, K. et al., Construction of mammarygland-specific expression vectors for human clotting factor IX and itssecretory expression in goat milk, Chin. J. Biotechnol. 13(4):271-6[1997]; Clark, A. J., Gene expression in the mammary glands oftransgenic animals, Biochem. Soc. Symp. 63:133-40 [1998]; Niemann, H. etal., Expression of human blood clotting factor VIII in the mammary glandof transgenic sheep, Transgenic Res. 8(3):237-47 [1999]).

Techniques for obtaining the preferred transgenic female mammalstypically employ transfection with an expression vector in which, withina transcriptional unit regulated, for example, by a suitableβ-lactoglobulin promoter, the PTTG-C peptide-encoding polynucleotide ischimerically linked with a polynucleotide encoding a mammary secretorysignal peptide, such that mammary-specific expression yields a chimericpolypeptide from which the desired PTTG-C peptide segment is removedproteolytically and purified.

The present invention is also directed to a kit for the treatment ofneoplastic cellular proliferation. The kit is useful for practicing theinventive method of inhibiting neoplastic cellular proliferation and/ortransformation. The kit is an assemblage of materials or components,including at least one of the inventive compositions containing aPTTG-C-related polynucleotide and/or PTTG-C peptides, as describedabove. The exact nature of the components configured in the inventivekit depends on its intended purpose. For example, some embodiments ofthe kit are configured for the purpose of treating cultured mammaliancells. Other embodiments are configured for the purpose of treatingmammalian cells in vivo, i.e., for treating mammalian subjects in needof treatment, for example, subjects with malignant tumors. In a mostpreferred embodiment, the kit is configured particularly for the purposeof treating human subjects.

Instructions for use are also included in the kit. “Instructions foruse” typically include a tangible expression describing the reagentconcentration or at least one assay method parameter, such as therelative amounts of reagent and sample to be admixed, maintenance timeperiods for reagent/sample admixtures, temperature, buffer conditions,and the like, typically for an intended purpose.

Optionally, the kit also contains other useful components, such as,diluents, buffers, pharmaceutically acceptable carriers, specimencontainers, syringes, stents, catheters, pipetting or measuring tools,paraphernalia for concentrating, sedimenting, or fractionating samples,or the inventive antibodies, and/or primers and/or probes for controls.

The materials or components assembled in the kit can be provided to thepractitioner stored in any convenient and suitable ways that preservetheir operability and utility. For example the components can be indissolved, dehydrated, or lyophilized form; they can be provided atroom, refrigerated or frozen temperatures.

The components are typically contained in suitable packagingmaterial(s). As employed herein, the phrase “packaging material” refersto one or more physical structures used to house the contents of thekit, such as invention nucleic acid probes or primers, and the like. Thepackaging material is constructed by well known methods, preferably toprovide a sterile, contaminant-free environment.

The packaging materials employed in the kit are those customarilyutilized in polynucleotide-based or peptide-based systems. As usedherein, the term “package” refers to a suitable solid matrix or materialsuch as glass, plastic, paper, foil, and the like, capable of holdingthe individual kit components. Thus, for example, a package can be aglass vial used to contain suitable quantities of an inventivecomposition containing nucleic acid or peptide components. The packagingmaterial generally has an external label which indicates the contentsand/or purpose of the kit and/or its components.

The invention will now be described in greater detail by reference tothe following non-limiting examples, which unless otherwise stated wereperformed using standard procedures, as described, for example inManiatis et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1982); Sambrooket al., Molecular Cloning: A Laboratory Manual (2 ed.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., USA (1989); Davis etal., Basic Methods in Molecular Biology, Elsevier Science Publishing,Inc., New York, USA (1986); or Methods in Enzymology: Guide to MolecularCloning Techniques Vol.152, S. L. Berger and A. R. Kimmerl Eds.,Academic Press Inc., San Diego, USA (1987).

EXAMPLES Example 1 Isolation of PTTG cDNA

To clarify the molecular mechanisms involved in pituitary tumorigenesis,differential display PCR was used to identify mRNAs differentiallyexpressed in pituitary tumor cells (see, e.g., Risinger et al., 1994,Molec. Carcinogenesis, 11:13-18; and Qu et al., 1996, Nature,380:243-247). GC and GH₄ pituitary tumor cell lines (ATCC #CCL-82 and#CCL-82.1, respectively) and an osteogenic sarcoma cell line UM108 (ATCC#CRL-1663) were grown in DMEM supplemented with 10% fetal bovine serum.Normal Sprague-Dawley rat pituitaries were freshly excised. Total RNAwas extracted from tissue cultured cells and pituitary tissue usingRNeasy™ kit (Qiagen) according to manufacturer's instructions. Trace DNAcontamination in RNA preparations was removed by DNase1 (GenHunterCorporation) digestion. cDNA was synthesized from 200 ng total RNA usingMMLV reverse transcriptase (GenHunter Corporation), and one of the threeanchored primers (GenHunter Corporation). The cDNA generated was used inthe PCR display.

Three downstream anchored primers AAGCT₁₁N (SEQ. ID. NO.:13; where N maybe A, G, or C), were used in conjunction with 40 upstream arbitraryprimers for PCR display. 120 primer pairs were used to screen mRNAexpression in pituitary tumors versus normal pituitary. One tenth of thecDNA generated from the reverse transcriptase reaction was amplifiedusing AmpliTaq DNA polymerase (Perkin Elmer) in a total volume of 20 μlcontaining 10 mM Tris, pH 8,4, 50 nM KCl, 1.5 nM MgCl₂, 0.001% gelatin,2 μM dNTPs, 0.2 μM each primer and 1 μl [³⁵S]dATP. PCR cycles consistedof 30 seconds at 94° C., 2 minutes at 40° C., and 30 seconds 72° C. for40 cycles. The products were separated on 6% sequencing gels, and driedgels were exposed to Kodak film for 24 to 48 hours.

After development, DNA fragments amplified from pituitary tumor andnormal pituitary were compared. Bands unique to pituitary tumor wereexcised from the gel, and DNA extracted by boiling in 100 μl water andprecipitated with ethanol in the presence of glycogen (GenHunterCorporation). DNA was reamplified using the original set of primers andthe same thermal cycling conditions except that the dNTP concentrationwas increased to 20 μM. Reaction products were run on 1% agarose gel andstained with ethidium bromide. Bands were excised from the gel, eluted(Qiagen), cloned in to TA vectors (Invitrogen) and sequenced usingsequenase (USB). Using 120primer pairs in the above-described PCR assay,11 DNA bands that appeared to be differentially expressed in pituitarytumor cells were identified. These bands were evaluated further byNorthern blot analysis, using the PCR products as probes.

For Northern blot analysis, 20 μg of total RNA were fractionated on 1%agarose gel, blotted on to nylon membrane and hybridized with randomprimed probe using Quickhyb solutions (Stratagene). After washing,membranes were exposed to Kodak films for 6 to 72 hours. As a result ofthe Northern blot assay, pituitary tumor specific signals were detectedfor 2 bands. DNA sequence analysis revealed that one sequence washomologous with Insulin-induced growth response protein, while theanother 396 base pair fragment (amplified using 5′-AAGCTTTTTTTTTTTG-3′[SEQ. ID. NO.:11] as the anchored primer and 5′-AAGCTTGCTGCTC-3′ [SEQ.ID. NO.:12] as an arbitrary primer) showed no homology to knownsequences in the GenBank. This 396 bp fragment detected a highlyexpressed mRNA of about 1.3 kb in pituitary tumor cells, but not innormal pituitary nor in osteogenic sarcoma cells.

Example 2 Characterization of cDNA Sequence Encoding PTTG

To characterize this pituitary tumor-specific mRNA further, a cDNAlibrary was constructed using mRNA isolated from rat pituitary tumorcells. Poly A+RNA was isolated from pituitary tumor GH₄ cells usingmessenger RNA isolation kit (Stratagene) according to manufacturer'sinstructions, and was used to construct a cDNA library in ZAP Expressvectors (Stratagene). The cDNA library was constructed using ZAPExpress™ cDNA synthesis and Gigapack III gold cloning kit (Stratagene)following manufacturer's instructions. The library was screened usingthe 396 bp differentially displayed PCR product (cloned into TA vector)as the probe. After tertiary screening, positive clones were excised byin vivo excision using helper phage. The resulting pBK-CMV phagemidcontaining the insert was identified by Southern Blotting analysis.Unidirectional nested deletions were made into the DNA insert usingEXOIII/Mung bean nuclease deletion kit (Stratagene) followingmanufacturer's instructions. Both strands of the insert DNA weresequenced using Sequenase (USB).

Using the 396 bp PCR fragment described in Example 1 as a probe, a cDNAclone of 974 bp (SEQ. ID. NO.:1) was isolated and characterized. ThiscDNA was designated as pituitary tumor-specific gene (PTTG). Thesequence of PTTG contains an open reading frame for 199 amino acids (SEQID NO:2). The presence of an in-frame stop codon upstream of thepredicted initiation codon indicates that PTTG contains the completeORF. This was verified by demonstrating both in vitro transcription andin vitro translation of the gene product as described in Example 3.

Example 3 In vitro Transcription and Translation of the PTTG

Sense and antisense PTTG mRNAs were in vitro transcribed using T3 and T7RNA polymerase (Stratagene), respectively. The excess template wasremoved by DNase I digestion. The in vitro transcribed mRNA wastranslated in rabbit reticular lysate (Stratagene). Reactions werecarried out at 30° C. for 60 minutes, in a total volume of 25 μlcontaining 3 μl in vitro transcribed RNA, 2 μl ³⁵S-Methionine (Dupond)and 20 μl lysate. Translation products were analyzed by SDS-PAGE (15%resolving gel and 5% stacking gel), and exposed to Kodak film for 16hours.

The results indicate that translation of in vitro transcribed PTTG sensemRNA results in a protein of approximately 25 KD on SDS-PAGE, whereas noprotein was generated in either the reaction without added mRNA or whenPTTG antisense mRNA was utilized.

Example 4 Northern Blot Analysis of PTTG mRNA Expression

A search of GenBank and a protein profile analysis (using a BLASTProgram search of databases of the national center for BiotechnologyInformation) indicated that PTTG shares no homology with knownsequences, and its encoded protein is highly hydrophilic, and containsno well recognized functional motifs. The tissue expression patten ofPTTG mRNA was studied by Northern Blot analysis. A rat multiple tissueNorthern blot was purchased from Clontech. Approximately 2 μg of polyA+RNA per lane from eight different rat tissues (heart, brain, spleen,lung, liver, skeletal muscle, kidney, and testis) was run on adenaturing formaldehyde 1.2% agarose gel, transferred to nylon membraneand UV-cross linked. The membrane was first hybridized to the fulllength PTTG cDNA probe, and was stripped and rehybridized to a humanβ-actin cDNA control probe. Hybridization was performed at 60° C. forone hour in ExpressHyb hybridization solution (Clontech). Washing wastwice 15 minutes at room temperature in2×SSC, 0.05%SDS, and twice 15minutes at 50° C. in 0.1% SSC, 0.1% SDS. Exposure time for PTTG probewas 24 hrs, and actin probe 2 hours.

The results of the Northern assay indicate that testis is the onlytissue, other than pituitary tumor cells, that expresses PTTG mRNA, andthe testis expression level is much lower (2 μg polyA+mRNA, 24 hourexposure) than in pituitary tumor cells (20 μg total RNA, 6 hourexposure). Interestingly, the testicular transcript (about 1 Kb) isshorter than the transcript in pituitary tumors (1.3 Kb), indicatingthat the mRNA is differentially spliced in testis, and that the 1.3 Kbtranscript is specific for pituitary tumor cells.

Example 5 Over-Expression of PTTG in NIH 3T3 Fibroblast Cells

Since PTTG mRNA is over-expressed in pituitary tumor cells, whether thisprotein exerts an effect on cell proliferation and transformation wasdetermined. An eukaryotic expression vector containing the entire codingregion of PTTG was stably transfected into NIH 3T3 fibroblasts.

The entire coding region of the PTTG was cloned in frame into pBK-CMVeukaryotic expression vector (Stratagene), and transfected into NIH 3T3cells by calcium precipitation. 48 hrs after transfection, cells werediluted 1:10 and grown in selection medium containing 1 mg/ml G418 fortwo weeks in when individual clones were isolated. Cell extracts wereprepared from each colony and separated on 15% SDS-polyacrylamide gels,and blotted onto nylon membrane. A polyclonal antibody was generatedusing the first 17 amino acids of PTTG as epitope (Research Genetics).The antibody was diluted 1:5000 and incubated with the above membrane atroom temperature for 1 hour. After washing, the membrane was incubatedwith horseradish peroxidase-labeled secondary antibody for one hour atroom temperature. The hybridization signal was detected by enhancedchemiluminescence (ECL detection system, Amersham).

Expression levels of the PTTG were monitored by immunoblot analysisusing the above-described specific polyclonal antibody directed againstthe first 17 amino acids of the protein. Expression levels of individualclones varied, and clones that expressed higher protein levels were usedfor further analysis.

Example 6 Effect of PTTG Expression on Cell Proliferation

A non-radioactive cell proliferation assay was used to determine theeffect of PTTG protein over-expression on cell proliferation (see, e.g.,Mosmann, T., 1983, J. Immunol. Meth., 65:55-63; and Carmichael et al.,1987, Cancer Res., 47:943-946). Cell proliferation was assayed usingCellTiter 96TM Non-radioactive cell proliferation assay kit (Promega)according to the manufacturer's instructions. Five thousand cells wereseeded in 96 well plates (6 wells for each clone in each assay), andincubated at 37° C. for 24 to 72 hours. At each time point, 15 μl of theDye solution were added to each well, and incubated at 37° C. for 4hours. One hundred μl of the solubilization/stop solution were thenadded to each well. After one hour incubation, the contents of the wellswere mixed, and absorbance at 595 nm was recorded using an ELISA reader.Absorbance at 595 nm correlates directly with the number of cells ineach well.

Three independent experiments were performed. The cell growth rate of3T3 cells expressing PTTG protein (assayed by cellular conversion oftetrazolium into formazan) was suppressed 25 to 50% as compared with 3T3cells expressing the pCMV vector alone, indicating that PTTG proteininhibits cell proliferation (data not shown).

Example 7 PTTG Induction of Morphological Transformation and Soft-agarGrowth of NIH 3T3 Cells

The transforming property of PTTG protein was demonstrated by itsability to form foci in manslayer cultures and showanchorage-independent growth in soft agar (Table 1). As primarypituitary cells are an admixture of multiple cell types and they do notreplicate in vitro, NIH 3T3 cells were employed. For the soft agar assay(Schwab et al., 1985, Nature, 316:160-162), 60 mM tissue culture plateswere coated with 5 ml soft-agar (20% 2× DEEM, 50% DEEM, 10% fetal bovineserum, 20% 2.5% agar, melted and combined at 45° C.). 2 ml cellssuspended in medium were then combined with 4 ml agar mixture, and 1.5ml of this mixture added to each plate. Cells were plated at a densityof 10⁴ cells/dish and incubated for 14 days before counting the numberof colonies and photography. Only colonies consisting of at least 40cells were counted. Values shown in Table 13 are means ± SEM oftriplicates.

TABLE 13 Colony Formation by NIH 3T3 Cells Transfected with PTTG cDNAConstructs Growth Efficiency of Colony Cell line in Soft Agar formationin Soft Agar (%)* No DNA 0 0   Vector only 1.3 ± 0.7  0.013 PTTG 3  26 ±4.6 0.26 PTTG 4 132 ± 26  1.32 PTTG 8  33 ± 6.0 0.33 PTTG 9 72 ± 13 0.72PTTG 10 92 ± 18 0.92 *Efficiency of colony formation was calculated aspercentage of number of colonies divided by total number of cells.

The results indicate that NIH 3T3 parental cells and 3T3 cellstransfected with pCMV vector do not form colonies on soft agar, whereas3T3 cells transfected with PTTG form large colonies. In addition, focaltransformation is observed in cells over-expressing PTTG protein, butcells expressing pCMV vector without the PTTG insert showed similarmorphology to the parental 3T3 cells.

Example 8 Assay to Determine whether PTTG is Tumorigenic In Vivo

To determine whether PTTG is tumorigenic in vivo, PTTG-transfected 3T3cells were injected subcutaneously into athymic nude mice. 3×10⁵ cellsof either PTTG or pCMV vector-only transfected cells were resuspended inPBS and injected subcutaneously into nude mice (5 for each group).Tumors were excised from sacrificed animals at the end of the 3rd weekand weighed. All injected animals developed large tumors (1-3 grams)within 3 weeks. The results are shown in Table 14 below. No mouseinjected with vector-only transfected cells developed tumors. Theseresults clearly indicate that PTTG is a potent transforming gene invivo.

TABLE 14 In vivo Tumorigenesis by NIH 3T3 Cells Transfected with PTTGcDNA Expression Vector Cell line No. Animals injected Tumor formationVector only 5 0/5 PTTG 4 5 5/5

Example 9 Human Carcinoma Cell Lines Express PTTG

The pattern of expression of PTTG in various human cell lines wasstudied employing a multiple human cancer cell line Northern blot(Clontech). The specific cell lines tested are shown in Table 15 below.

TABLE 15 Human Carcinoma Cell Lines Tested Cell Line PTTG Expression 1Promyelocytic Leukemia HL-60 + 2 HeLa Cell S3 + 3 Chronic MyelogenousLeukemia K-562 + 4 Lymphoblastic Leukemia MOLT-4 + 5 Burkilt's lymphomaRaji + 6 Colorectal Adenocarcinoma SW 480 + 7 Lung Carcinoma A549 + 8Melanoma G361 +

About 2 μg polyA RNA from each of the 8 cell lines indicated in Table 3above were placed on each lane of a denaturing formaldehyde 1.2% agarosegel, separated by denaturing gel electrophoresis to ensure intactness,transferred to a charge-modified nylon membrane by Northern blotting,and fixed by UV irradiation. Lanes 1 to 8 contained RNA frompromyelocytic leukemia HL-60, HeLa cell line S3, human chronicmyelogenous leukemia K-562, lymphoblastic leukemia MOLT-4, Burkitt'slymphoma Raji, colorectal adenocarcinoma SW 480, lung carcinoma A549 andmelanoma G361, respectively. RNA size marker lines at 9.5, 7.5, 4.4,2.4, and 1.35 kb were indicated in ink on the left margin of the blot,and utilized as sizing standards, and a notch was cut out from the lowerleft hand corner of the membrane to provide orientation. Radiolabeledhuman β-actin cDNA was utilized as a control probe for matching ofdifferent batches of polyA RNAs. A single control band at 2.0 kb in alllanes spotted is confirmatory.

The blots were probed with the full length rat PTTG cDNA probe (SEQ. IDNo: 1; 974 bp) at 60° C. for 1 hr. in ExpressHyb hybridization solution(Clontech) as described by Sambrook et al., the relevant section ofwhich reference is incorporated herein by reference. See, Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd. Ed, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). The blots were thenwashed twice for 15 min at room temperature in 2×SSC, 0.05% SDS, andtwice for 15 min at 50° C. in 0.1% SSC, 0.1% SDS. A more detaileddescription of the remaining experimental procedures masy be found inPei & Melmed, the relevant section of which is incorporated herein byreference. (See, Pei & Melmed, Endocrinology 4: 433-441 [1997]).

All cells tested by the Northern blot analysis as described aboveevidenced expression of human PTTG (i.e., PTTG1), including lymphoma,leukemia, melanoma and lung carcinomas, among others.

Example 10 Molecular Cloning of Human PTTG cDNA

A human fetal liver cDNA library (Clontech, Palo Alto, Calif.) wasscreened as described by Maniatis et al. (Maniatis et al., Molecularcloning, Cold Spring Harbor Press, 1989), using a radioactively labeledcDNA fragment of the entire rat PTTG coding region as a probe. The cDNAinserts from positive clones were subcloned into plasmid pBluescript-SK(Stratagene, La Jolla, Calif.), and subjected to sequence analysis usingSequenase kit (U.S. Biochemical Corp., Cleveland, Ohio).

A complete open reading frame containing 606 bp was found in thepositive clones. The homology between the nucleotide sequences of theopen reading frame and the coding region of rat PTTG is 85%. Amino acidsequence comparison between the translated product of this open readingframe and rat PTTG protein reveals 77% identity and 89% homology. ThecDNAs obtained from these clones represents human homologies of ratPTTG. No other cDNA fragments with higher homology were detected fromthe library.

Example 11 Tissue Distribution of Human PTTG mRNA

Total RNA was prepared using Trizol Reagent (Gibco-BRL, Gaithersburg,Md.) from normal human pituitary glands (Zoion Research Inc. Worcester,Mass.) and fresh human pituitary tumors collected at surgery and frozenin liquid nitrogen. 20 mg total RNA were used for 1% agarose gelelectrophoresis. RNA blots (Clontech, Palo Alto, Calif.) derived fromnormal adult and fetal tissues as well as from malignant tumor celllines, were hybridized with radioactively labeled human cDNA fragmentcontaining the complete coding region. The RNA isolated from each cellline was transferred onto a nylon membrane (Amersham, Arlington Heights,Ill.), and hybridized with radioactively labeled probe at 55° C.overnight in 6×SSC, 2× Denhardt's solution, 0.25% SDS.

The membranes were washed twice at room temperature for 15 minutes each,and then for 20 minutes at 60° C. in 0.5×SSC, 0.1% SDS, andautoradiographed. The autoradiography was carried out using KodakBIOMEX-MR film (Eastman Kodak, Rochester, N.Y.) with an intensifyingscreen. The blots were stripped by washing for 20 minutes in distilledwater at 95° C. for subsequent probing.

The results from the Northern blot analysis indicated that PTTG isexpressed in liver, but not in brain, lung, and kidney of human fetaltissue. In addition, PTTG is strongly expressed in testis, modestlyexpressed in thymus, and weakly expressed in colon and small intestineof normasl human adult tissue. No expression was detected by Northernanalysis in brain, heart, liver, lung, muscle, ovary, placenta, kidney,and pancreas.

The expression of PTTG in several human carcinoma cell lines was alsoanalyzed by Northern blots. In every carcinoma cells examined, PTTG wasfound highly expressed. The human tumor cell lines tested are listed inTable 16 below.

TABLE 16 Tested Human Tumor Cell Lines Promyelocytic leukemia HL-60Epitheloid carcinoma HeLa cell S3 Chronic myelogenous leukemia K-562Lymphoblastic leukemia MOLT-4 Burkitt's lymphoma Raji Colorectaladenocarcinoma SW 480 Lung carcinoma A549 Melanoma G361 Hepatocellularcarcinoma Hep 3B Thyroid carcinoma TC-1 Breast adenocarcinoma MCF-7Osteogenic sarcoma U2 OS Placenta choriocarcinoma JAR ChoriocarcinomaJEG-3

Example 12 Human PTTG Expression in Normal Pituitary and PituitaryTumors

RT-PCR was performed as follows. 5 mg total RNA were treated with 100 URNase-free DNase I at room temperature for 15 minutes. DNase I wasinactivated by incubation at 65° C. for 15 minutes. The sample was thenused for reverse transcription using oligo-dT primer and SuperScript IIreverse transcriptase (Gibco-BRL, Gaithersburg, Md.). After reversetranscription, the sample was subjected to PCR amplification with PCRSuperMix (Gibco-BRL, Gaithersburg, Md.) using hPTTG-specific primers andhuman cyclophilin A-specific primers as an internal control.

Northern blot analysis indicated that the level of expression of PTTG isquite low in normal pituitary as well as in pituitary tumors. Therefore,comparative RT-PCR was used to study the expression of PTTGquantitatively in normal pituitary and pituitary tumors. The results ofthis study showed that in most of pituitary tumors tested, includingnon-functioning tumors, GH-secreting tumors, and prolactinomas, theexpression level of PTTG was higher than that of normal pituitary.

Example 13 Stable Transfection of Human PTTG into NIH 3T3 Cells

The complete coding region of hPTTG cDNA was subcloned in reading frameinto the mammalian expression vector pBK-CMV (Stratagene, La Jolla,Calif.), and transfected into NIH 3T3 fibroblast cells by Lipofectamine(Gibco-BRL, Gaithersburg, Md.) according to manufacturer's protocol. 24hours after transfection, the cells were serially diluted and grown inselection medium containing 1 mg/ml G418 for 2 weeks. Individual cloneswere isolated and maintained in selection medium. Total RNA was isolatedfrom hPTTG-transfected cell lines as well as from control cells in whichblank vector pBK-CMV had been transfected. Northern blot was performedto confirm overexpression of hPTTG in transfected cell lines. These celllines were used in subsequent cell proliferation assay as well as invitro and in vivo transformation assay.

Example 14 Cell Proliferation Assay

A cell proliferation assay was performed using the CellTiter 96non-radioactive cell proliferation assay kit (Promega Medicine, Wis.)according to the manufacturer's protocol. 5,000 cells were seeded in96-well plates and incubated at 37° C. for 24-72 hours. Eight wells wereused for each clone in each assay. At each time point, 15 ml of dyesolution was added to each well and the cells were incubated at 37° C.for 4 hours. After incubation, 100 ml solubilization/stop solution wereadded to each well, and the plates incubated overnight at roomtemperature. The absorbance was determined at 595 nm using an ELISAplate reader.

Control and hPTTG-overexpressing NIH 3T3 cells were used to perform thisassay. The results indicated that the growth of cells transfected withthe PTTG-expressing vector was suppressed by 30˜45% as compared withcells transfected with blank vector. These results clearly show that thePTTG protein inhibits cell proliferation.

Example 15 In Vitro and In Vivo Transformation Assay

(a) In vitro transformation assay

Control and hPTTG-transfected cells were tested foranchorage-independent growth in soft agar. 3 ml of soft agar (20% of 2×DMEM, 50% DMEM, 10% fetal bovine serum, and 20% of 2.5% agar, melted andmixed at 45° C.) were added to 35 mm tissue dishes. 10,000 cells weremixed with 1 ml soft agar and added to each dish, and incubated for 2weeks until colonies could be counted and photographed.

(b) In vivo transformation assay

5×10⁵ cells containing either a blank vector or hPTTG-expressing cellswere injected into nude mice. The mice were sacrificed two weeks afterinjection, and the tumors formed near the injection sites examined.

When the NIH 3T3 cells stably transfected with the PTTG-expressingvector were tested in an anchorage-independent growth assay, these cellscaused large colony formation on soft agar, suggesting the transformingability of PTTG protein.

When the NIH 3T3 cells were injected into nude mice, they caused in vivotumor formation within 2 weeks after injection. These data indicate thathuman PTTG, as its rat homologue, is a potent transforming gene.

Example 16 Inhibition of Cell Transformation/Tumor Formation by PTTGC-Terminal Polypeptide

Cell lines. NIH 3T3 cells were maintained in high glucose (4.5 g/L) DMEM(Gibco-BRL) supplemented with 10% fetal bovine serum. HeLa cells weremaintained in low glucose (1 g/L) DMEM (Gibco-BRL) supplemented with 10%fetal bovine serum (FBS). T-47D and MCF-7 cells were maintained in highglucose DMEM (Gibco-BRL) supplemented with 10% fetal bovine serum and0.01 mg/mL bovine insulin (Sigma). All cell lines were obtained fromAmerican Type Culture Collection (ATCC).

Site-directed mutagenesis and stable transfection of human and mutantPTTG into NIH 3T3 cells. Point mutations on the proline-rich domain(s)of wild type human PTTG polypeptide (wtPTTG) were generated by PCR-basedsite-directed mutagenesis. Two synthetic oligonucleotides,5′-GATGCTCTCCGCACTCTGGGAATCCAATCTG-3′ (SEQ. ID. NO.:5) and5′-TTCACAAGTTGAGGGGCGCCCAGCTGAAACAG-3′ (SEQ. ID. NO.:6), which causepoint mutations that result in amino acid sequence changes P163A, S165Q,P166L, P170L, P172A, and P173L in the wtPTTG protein, were used toamplify human PTTG cDNA cloned into pBlue-Script-SK vector (Stratagene).Amplified mutated cDNA (mutPTTG) was then cloned into mammalianexpression vector pCI-neo (Promega). Overexpression of mutPTTG intransfected cells was confirmed by Northern analysis and RT-PCR followedby direct sequence analysis. wtPTTG and mutPTTG were subcloned intopCl-neo, and the vector was used to transfect NIH 3T3 cells as describedin Zhang, X., et al. [1999a].

Transactivation assay. wtPTTG cDNA was fused in frame with pGAL4(Stratagene), designated pGAL4-wtPTTG and was used as template fordeletion and mutation analysis; mutPTTG cDNA was also fused in framewith pGAL4 and designated pGAL4-mutPTTG. pGAL4-VP 16 was used as apositive control. Experimental plasmids; were co-transfected with pLUCand pCMV-β-Gal (as internal control). Cell lysates were prepared 48hours after transfection and assayed for luciferase activity asdescribed (Wang, Z. and Melmed, S. [2000]; Sambrook, J., et al.,Molecular Cloning: A Laboratory Manual, 2d Ed. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y., 10.2.1-10.2.6[1989]).

Constructions of expression vectors for wild type and mutant human PTTGC-terminal polypeptides. To generate wtPTTG and mutPTTG C-terminalpolypeptide expression vector, the internai Xba I site of wtPTTG andmutPTTG cDNA and the 3 ′-portions of these cDNAs were cloned intopCI-neo (Promega, Madison, Wis.) via Xba I and Not I sites. In theseclone, the ATG for M147 of full-length PTTG is used as an initiationcodon, generating a polypeptide of 56 amino acid residues correspondingto nucleotide positions 147 through 202 of full-length wtPTTG.

Stable transfection of human PTTG C-terminal peptide into tumor cells.Wild type and mutant PTTG C-terminal expression constructs weretransfected into HeLa, MCF-7, and T47-D cells with Lipofectin(GIBCO-BRL) according to the manufacturer's protocol. Twenty-four hoursafter transfection, cells were serially diluted and selected with G418(1 mg/ml) for 2 weeks. Individual clones were isolated and maintained inselection medium (respective high or low glucose DMEM with 10% FBS, asdescribed above, and G148 [1 mg/mL]), and total RNA was extracted fromtransfected cells. Expression of wild-type and mutated PTTG -C terminalwas confirmed by RT-PCR using two synthetic oligonucleotides, with onespecific to the 5′-nontranslational region from vector pCI-neo,5′-GGCTAGAGTACTTAATACGACTCACTATAGGC-3′ (SEQ. ID. NO.:7), and the otherto the 3′-translational region of PTTG1 cDNA,5′-CTATGTCACAGCAAACAGGTGGCAATTCAAC-3′ (SEQ. ID. NO.:8), followed bydirect sequence analysis.

In vitro colony formation and in vivo tumorigenesis. NIH 3T3 stabletransfectants were tested in vivo as described in Zhang, X., et al.[1999a]. Transfected cells were tested for anchorage-independent growthin soft agar as described Zhang, X., et al. [1999a]. HeLa cells wereincubated for 3 weeks and MCF-7 and T-47D cells for 2 weeks. For in vivoassays of tumorigenesis, 1×10⁷ MCF-7 stable transfectants wereresuspended in 500 μL MATRIGEL basement membrane matrix (BectonDickinson, Bedford, Mass.) and were injected subcutaneously into nudemice (three mice for each group). After four weeks, animals werephotographed and tumors were excised and weighed.

ELISA of basic fibroblast growth factor (bFGF) in conditioned medium.The concentration of basic fibroblast growth factor (bFGF) concentrationin HeLa cell culture medium was assayed using Quantikine HS Human FGFBasic Immunoassay Kit (R&D Systems, Minneapolis, Minn.) according to themanufacturer's protocol. Cells (1×10⁵) were plated in 100-mm cellculture dishes. After 72 hours, the culture medium was collected and 200mL was used for ELISA assay.

Effects of wild type human PTTG and mutant PTTG overexpression on tumorinduction. It was previously demonstrated that NIH 3T3 cellsoverexpressing wild type PTTG formed large colonies in ananchorage-independent growth assay and formed tumors when injected intoathymic nude mice, while point mutations in the proline-rich region(P163A, P170L, P172A, and P173L) abrogated formation of colonies andtumors (Zhang, X., et al. [1999a]). Overexpression of wtPTTG and mutPTTG(P163A, S165Q, P166L, P170L, P172A, and P173L) in each transfectant cellline was confirmed by Northern analysis and RT-PCR followed by directsequence analysis (not shown).

It was further shown that overexpressing PTTG transfectants injectedinto athymic nude mice caused tumor formation within 2 weeks in allinjected animals. Five mice in each of three groups were injectedsubcutaneously with 3×10⁵ NIH 3T3 cells transfected with: (1) controlcell line (transfected with pGAL4 vector alone); (2) wild typePTTG-overexpressing (wtPTTG); or (3) mutant PTTG-overexpressing (mutPTTG[P163A, S 165Q, P166L, P170L, P172A, and P173L]). After 2 weeks, micewere sacrificed and tumors were excised and weighed. In the miceinjected with control transfectants or mutPTTG transfectants, no tumorsdeveloped, but mice injected with transfectant cells bearing wtPTTGdeveloped tumors without exception. Tumor weights ranged from 470 to1500 mg (Table 17).

TABLE 17 Tumor formation by PTTG-expressing NIH 3T3 Cells in AthymicNude Mice. Tumor weight (mg) Vector wtPTTG mutPTTG  none* 1500 none none 770 none none 1250 none none  550 none none  470 none *none = nodetectable tumor.PTTG exhibits transcriptional activation. Vector pGal4 alone (negativecontrol) did not activate the luciferase (luc) reporter, and a knownactivation domain, VP16, significantly increased reporter activity about28-fold. pGAL4-wtPTTG exhibited transactivation properties and inducedreporter activity about 22-fold (FIG. 1).

Transcriptional activity of pGAL4-mutPTTG (mutated proline region[P163A, S165Q, P166L., P170L, P172A, and P173L]), other point mutations,as well as a separate deletions (d) of wtPTTG were also tested asindicated in Table 18. In Table 18, the indicated plasmids wereco-transfected with pLuc and pCMV-β-Gal into NIH 3T3 cells, andluciferase assays were performed, with β-Gal serving as the internalcontrol. Each value represents triplicate wells from two independentexperiments (±SEM); transactivation by wtPTTG was designated 100%.pGAL4-mutPTTG exhibited about 95% transactivating activity compared topGAL4-wtPTTG, thus confirming the importance of the wtPTTG proline-richmotif for transactivation.

TABLE 18 Transactivation assay of hPTTG mutants. Mutant activationactivity (%) (± SEM) pGAL4-wtPTTG 100 -163 Pro → Ala 100 ± 10 -166 Pro →Ala  45 ± 5* -170 Pro → Ala 100 ± 10 -182 Pro → Ala 100 ± 10 -152 Glu →Gln 100 ± 10 -192 Glu → Gln  50 ± 3* -165 Ser → Ala  30 ± 3* -165 Ser →Leu  20 ± 2* -176 Ser → Ala 100 ± 10 -183 Ser → Ala 100 ± 10 -184 Ser →Ala 100 ± 10 -d(1-100) 100 ± 10 -d(180-202) 100 ± 10 -mutPTTG  6 ± 1* *p< 0.01Human PTTG C-terminal peptide expression blocks cell transformation. Thecritical role of the proline-rich region in transactivation,transformation and tumor formation, as described above, implies thatPTTG functions through SH3-mediated signal transduction. If human PTTG1protein mediates the SH3 -related signal cascade, it probably containsat least two functional domains interacting with upstream and downstreamsignal molecule(s), respectively. A mutant protein containing only onesuch functional domain could then act in a dominant-negative manner toabrogate wild-type protein function and disrupt signal transduction.

Based on this hypothesis, a truncated PTTG1 mutant peptide, lackingN-terminal amino acid residues 1-146, was introduced into humancarcinoma cells. An expression construct was used expressing a PTTG-Cpeptide corresponding to residues 147-202 of the full-length protein,under the control of a CMV promoter. This polypeptide contains theproline-rich domain(s) (residues 163-173; Zhang, X., et al. [1999a]),and when the coding sequence was fused to glutathione S-transferase(GST), it was expressed in Escherichia coli as an intact protein withthe appropriate molecular weight (data not shown). Mutant expressionvector pCIneo-mutPTTG (mutated proline region [P163A, S165Q, P166L.,P170L, P172A, and P173L]), as well as the empty vector pCI-neo alone ascontrol, were stably transfected into HeLa, MCF-7, and T-47D humancarcinoma cell lines.

Transfectants expressing wild-type PTTG carboxy-terminal peptide(PTTG-C), PTTG C-terminal mutated in several proline residues (PTTG-Cpm;mutated proline region [P163A, S165Q, P166L., P170L, P172A, and P173L]),and vector (V), were isolated. Expression of each transfectant line wasconfirmed by RT-PCR, using a primer directed to the 5′-nontranslationalregion of the expression vector and a primer directed to the3′-translational region of PTTG mRNA, followed by direct sequenceanalysis (FIGS. 2A, 2B, and 2C). Transforming abilities of all three ofthese stably transfected cell lines were tested in ananchorage-independent growth assay, PTTG-Cpm cells were observed to formlarge colonies, as did control V cells containing the same expressionvector but lacking either wild type or mutant C-terminal polypeptide.Each transfectant cell line was plated in three different plates. HeLawas scored on the 21st day and T-47D and MCF-7 on the 14th day. Coloniesconsisting of 60 or more cells were scored. However, the number andsize, of colonies formed by cells expressing PTTG-C were markedlyreduced (p<0.01) (FIG. 3). Table 19 (below) summarizes the soft agarcolony formation for each cancer cell type.

TABLE 19 Colony Formation by PTTG I C-terminal (PTTG-C) and mutant PTTGC-terminal (PTTG-Cpm) Expressing Cells in Soft Agar. Colonies/10⁴ CellsCell Line Vector (mean ± SEM) HeLa Vector alone 1465 ± 54  Vector alone2392 ± 55  PTTG-C 11 ± 2* PTTG-C  6 ± 1* PTTG-C 48 ± 3* PTTG-C  3 ± 1*PTTG-Cpm 1169 ± 77  PTTG-Cpm 1097 ± 79  PITG-Cpm 2615 ± 76  T-47D Vectoralone 135 ± 4  PTTG-C 46 ± 5* PTTG-C 52 ± 2* PTTG-Cpm 193 ± 5  PTTG-Cpm106 ± 5  MCF-7 Vector alone 287 ± 3  PTTG-C  9 ± 3* PTTG-C 34 ± 4*PTTG-Cpm 236 ± 11  PTTG-Cpm 206 ± 4  *P < 0.01

Human PTTG C-terminal polypeptide-expressing MCF-7 cells fail to developtumors in vivo. Stably transfected MCF-7 cell lines were injected (1×10⁷cells/per mouse in 500 μL MATRIGEL basement membrane matrix)subcutaneously into athymic nude mice. After four weeks, mice werephotographed, killed, and their tumors were excised and weighed. Threemice injected with cells transfected with control vector only developedvisible tumors in 4 weeks, while three mice injected withPTTG-C-transfected cells failed to generate tumors. At autopsy, absenceof subcutaneous or other peripheral tumor formation was confirmed in themice receiving PTTG-C transfected cells. Three mice injected withPTTG-Cpm-transfected cells also developed tumors after 4 weeks, whichwere similar in size to those developed in mice injected with controlvector-transfected cells, indicating that the mutated PTTG-C-terminalpolypeptide lost its ability to abrogate endogenous PTTG function (Table20).

TABLE 20 Tumor formation by PTTG-C expressing MCF-7 Cells in AthymicNude Mice. Tumor weight (mg) Vector PTTG-C PTTG-Cpm 212  none* 185 235none 196 209 none 203 *none = no detectable tumor.

These results show that overexpression of the PTTG C-terminal peptidecaused cancer cells to lose their abilities for in vitro celltransformation and ex vivo tumor growth. Also, the importance ofproline-rich regions is further confirmed here, since PTTG C-terminalpeptide containing point mutations of these proline residues failed tointerfere with transforming activity or tumor-forming activity in vivo.

Suppression of bFGF secretion and PRL expression by PTTG-C peptide. Ascells expressing wild-type human PTTG-C terminal peptide had markedlyreduced colony forming ability on soft agar and were also unable toinduce solid tumor growth in vivo, expression of bFGF was tested in HeLatransfectants. An enzyme-linked immunoabsorbent assay (ELISA) wasperformed to examine bFGF levels in conditioned medium derived from72-hour cultures of HeLa transfectants. As shown in FIG. 4, bFGF levelswere markedly decreased in conditioned medium derived from PTTG-CDNA-transfected cells than those derived from vector-only andPTTG-Cpm-transfected cells, indicated a suppression of bFGF secretionresulting from the presence of PTTG carboxy-terminal peptide.

Since, the growth rate of solid tumors is directly related to activationof angiogenesis and recruitment of new blood vessels, this shows that,in accordance with the inventive method, the ability for new bloodvessel growth can be impaired by the inventive PTTG-C peptides,providing an additional mechanism leading to the failure of in vivoneoplastic cellular proliferation and tumor growth. Experimental tumorsdo not grow more than 1 or 2 mm in diameter in the absence ofangiogenesis. (Folkman, J., N. Engl. J. Med. 285:1182-1186 [1971];Folkman, J., and Klagsburn, M. (1987) Science 235:442-447 [1987]). Thehuman cancer cell lines used in this study form prominent solid tumors(>2 mm in diameter) indicating active angiogenesis.

Moreover, these results imply that additional hormonal regulatorycascades can be affected by the inventive PTTG-C peptides, becausereduced bFGF secretion can result in altered expression of bFGF-mediatedpathways, for example prolactin (PRL) expression. For example,expression of the same human wild-type PTTG-C-terminal peptide (aminoacid residues 147-202 of SEQ. ID. NO.:4) in rat prolactin (PRL)- andgrowth hormone (GH)-secreting GH3 cells caused markedly reduced PRLpromoter activity (about 16-fold decrease), PRL mRNA expression (about10-fold decrease), and prolactin protein expression (about 72-folddecrease) in comparison to rat GH3 cells transfected with control vectoralone or GH3 cells expressing a mutated PTTG1 C-terminal fragment(P163A, S165Q, P166L, P170A, P172A, and P173L; data not shown).Furthermore, a compensatory increase in GH mRNA (about 13-fold increase)and protein (about 37-fold increase) were observed in thePTTG-C-terminal expressing GH3 cells. These observations demonstratethat PTTG carboxy-terminal peptide expressed in GH3 cells alters thehormonal secretory pattern by silencing PRL-gene expression andaugmenting GH expression.

1. A method of inhibiting neoplastic cellular proliferation and/ortransformation of a cell of mammalian origin comprising: delivering to amammalian cell that expresses PTTG a composition comprising a PTTG-Cpeptide consisting of SEQ ID NO: 9, complexed with a cellularuptake-enhancing agent, in an amount and under conditions sufficient toenter the cell, whereby neoplastic cellular proliferation and/or frameformation of the cell is inhibited.
 2. The method of claim 1, whereinthe cell is of human origin.
 3. The method of claim 1, wherein the cellexhibits neoplastic, hyperplastic, cytologically dysplastic, orpremalignant cellular growth or proliferation.
 4. The method of claim 1,wherein the cell is a malignant cell.
 5. The method of claim 1, whereinthe composition is delivered to the cell in vitro.
 6. The method ofclaim 1, further comprising administering the composition to a mammaliansubject, such that the PTTG-C peptide is delivered to the cell in vivo.7. The method of claim 1, wherein said uptake enhancing agent comprisesa polycationic lipid.
 8. The method of claim 1, wherein said uptakeenhancing age t comprises a cellular uptake-enhancing and/orimportation-competent peptide segment.
 9. The method of claim 8, whereinthe cellular uptake-enhancing and/or importation-competent peptidesegment is a human immunodeficiency virus TAT-derived peptide segment ora signal peptide from Kaposi fibroblast growth factor.
 10. An isolatedprotein, consisting of: a PTTG carboxy-terminal peptide consisting ofSEQ ID NO:9.
 11. A composition for inhibiting neoplastic cellularproliferation and/or transformation in a cell that expresses PTTG,comprising the protein of claim 10 and a pharmaceutically acceptablecarrier.
 12. The composition of claim 11, further comprising a cellularuptake-enhancing agent complexed with said PTTG carboxy-terminalpeptide.
 13. The composition of claim 11, wherein said uptake enhancingagent comprises a lipid agent.
 14. The composition of claim 12, whereinsaid uptake enhancing agent comprises a polycationic lipid agent. 15.The composition of claim 12, wherein said uptake enhancing agentcomprises a cellular uptake-enhancing and/or importation-competentpeptide segment.
 16. The composition of claim 15, wherein the cellularuptake-enhancing and/or importation-competent peptide segment is a humanimmunodeficiency virus TAT-derived peptide segment or a signal peptidefrom Kaposi fibroblast growth factor.
 17. A chimeric protein, consistingof: (i) a first segment consisting of a PTTG carboxy-terminal peptideconsisting of SEQ ID NO:9; and (ii) a second segment, which is acellular uptake-enhancing and/or importation-competent peptide segment.18. A composition for inhibiting neoplastic cellular proliferationand/or transformation in a cell that expresses PTTG, comprising thechimeric protein of claim 17 and a pharmaceutically acceptable carrier.19. The composition of claim 18, wherein the cellular uptake-enhancingand/or importation-competent peptide segment is a human immunodeficiencyvirus TAT-derived peptide segment or a signal peptide from Kaposifibroblast growth factor.
 20. A kit for the treatment of neoplasticcellular proliferation in cells that express PTTG, said kit comprising:the composition of claim 11; and instructions for the use of saidcomposition for inhibiting neoplastic cellular proliferation and/ortransformation in cells that express PTTG.
 21. A kit for the treatmentof neoplastic cellular proliferation in cells that express PTTG, saidkit comprising: the composition of claim 15; and instructions for theuse of said composition for inhibiting neoplastic cellular proliferationand/or transformation in cells that express PTTG.
 22. A kit for thetreatment of neoplastic cellular proliferation in cells that expressPTTG, said kit comprising: the composition of claim 18; and instructionsfor the use of said composition for inhibiting neoplastic cellularproliferation and/or transformation in cells that express PTTG.